Targeted delivery system and methods of use therefor

ABSTRACT

Disclosed are peptides and peptidomimetics that in some embodiments include the amino acid sequence KRGARST or (SEQ ID NO: 1), AKRGARSTA or (SEQ ID NO: 2), or CKRGARSTC (SEQ ID NO: 3). Also disclosed are conjugates and compositions that onclude the peptides and/or peptidomimetics, methods for directing a moiety to tumor lymphatic vasculature, methods for imaging tumor lymphatic vasculature, methods for reducing or inhibiting tumor metastasis, methods for reducing the number of tumor lymphatic vessels, methods for treating cancer, methods for treating a disease or disorder associated with a gC1q/p32 receptor biological activity, methods for detecting the presence of a gC1q/p32 receptor, methods for detecting interactions between gC1q/p32 receptors and the presently disclosed conjugates and compositions, methods for delivering the presently disclosed conjugates and compositions to gC1q/p32 receptors, methods for assessing gC1q/p32 receptor levels in cells, methods for identifying subjects having diseases associated with gC1q/p32 receptor biological activities, and methods for screening for compounds that interact with gC1q/p32 receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims the benefit of U.S. ProvisionalPatent Application Ser. Nos. 62/151,674 and 62/151,703, both filed Apr.23, 2015. The disclosure of each of these applications is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants CA183287,CA167174, CA152327, and CA121949 from the National Institutes of Health.The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted with the instant application as a ASCIItext file entitled “1861_31_ST25.txt” created on Apr. 16, 2015 andhaving a size of 114 kilobytes is hereby incorporated by reference inits entirety.

TECHNICAL FIELD

The present invention relates generally to the fields of molecularmedicine and cancer biology and, more specifically, to molecules thatinteract with the gC1q/p32 receptor.

BACKGROUND

The gC1q/p32 receptor is a mitochondrial chaperone protein responsiblefor the maintenance of certain proteins in the mitochondrial oxidativephosphorylation machinery (Fogal et al., 2010; Yagi et al., 2012). Thisprotein is also component of the CI complex of the classical complementpathway (Sim & Reid, 1991). The biological functions of the gC1q/p32receptor are diverse, including initiation of the complement cascade foropsonization and cytolysis, and mediation of several different functionsdepending on the cell types expressing the gC1q/p32 receptor. ThegC1q/p32 receptor enhances FcR and CR1-mediated phagocytosis inmonocytes/macrophages (Bobak et al., 1987; Bobak et al., 1988);stimulates immunoglobulin production by B cells (Young et al., 1991);activates platelets to express αIIb/β3 integrins, P-selectin, andprocoagulant activity (Peerschke et al., 1993; Peerschke et al., 1994);activates tumor cytotoxicity of macrophages (Leu et al., 1990); exertsanti-proliferative effects on T cell growth (Chen et al., 1994); andserves as a receptor for the Listeria monocytogenes invasion proteinInIB (Braun et al.,).

A 33 kilodalton (kDa) receptor, designated gC1qR/p32 (and alternativelyreferred to herein as p32, gC1q-R, or the gC1/p32 receptor), has beenidentified, cloned, and sequenced (Chen et al., 1994; Ghebrehiwet etal., 1994; Peerschke et al., 1994). The crystal structure of gC1qR/p32has also been solved (Jiang et al., 1999). Another 60 kDa receptor,designated cC1qR, binds to the amino-terminal collagen-like region ofC1q (Ghebrehiwet, 1989; Chen et al., 1994). Based on the detection ofgC1qR/p32 mRNA by polymerase chain reaction (PCR) amplification andgC1qR/p32 protein expression by immunochemical methods, this receptorwas found to exist on a large number of different cell types, such asbut not limited to B cells, T cells, monocytes/macrophages, neutrophils,eosinophils, fibroblasts, platelets, endothelial cells, liver cells,neural cells, and smooth muscle cells. The gC1q-R protein isover-expressed in tumor cells and tumors (Rubinstein et al., 2004).

The endothelial lining of blood vessels is highly diversified. Many, andperhaps all, normal tissues impart a tissue-specific “signature” ontheir vasculature, and tumor vessels differ from normal vessels both inmorphology and molecular composition (Ruoslahti, 2002). Tumors induceangiogenesis to support expansive growth (Hanahan & Weinberg, 2000 andmany of the changes in tumor vessels are angiogenesis related (Brooks etal., 1994; Ferrara et al., 1999; Pasqualini et al., 2000; Christian etal., 2003). Moreover, tumor blood vessels have tumor type-specific and,in some stages, stage-specific characteristics; in vivo screening ofphage libraries has yielded distinct sets of homing peptides selectivelyrecognizing angiogenic signatures in two transgenic mouse models oforgan-specific tumorigenesis. Homing peptides can also distinguish theangiogenic blood vessels of premalignant lesions from those of fullymalignant lesions in the same tumor. Lymphatic vessels in tumors alsocarry specific markers that distinguish tumor lymphatics from lymphaticsin normal tissues (Laakkonen et al., 2002; Laakkonen et al., 2004; Zhanget al., 2006). Tumor blood vessels and lymphatics provide importanttargets for tumor therapy. Destroying tumor blood vessels or preventingtheir growth suppresses tumor growth, whereas tumor lymphatics are notessential for tumor growth, but destroying them reduces metastasis(Saharinen et al., 2004).

The gC1qR/p32 protein is primarily a mitochondrial protein, but it isalso expressed at the cell surface. Its expression is greatly increasedin many cancers, particularly in breast cancer, and in atheroscleroticlesions. More importantly, the expression of p32 is specific for tumorcells and cells in atherosclerotic plaques at the level of cell surfaceexpression, which of p32 is a characteristic of cells in theseconditions, and not detectable in p32-expressing normal cells. Inaddition to tumor cells, a macrophage population associated with tumorlymphatics expresses high levels of total and cell-surface p32. p32expression is primarily found in poorly vascularized,hypoxic/nutrient-deprived regions, which are not readily accessible toconventional therapies.

A peptide that binds to p32 at the cell surface and inhibits tumorgrowth upon systemic administration called LyP-1 has been identified.LyP-1 (CGNKRTRGC; SEQ ID NO: 7) accumulates in tumors andatherosclerotic plaques, where it primarily accumulates in activatedmacrophage/myeloid lineage cells (Hamzah et al., 2011). The homing ofLyP-1 to these lesions is specific; LyP-1 does not accumulate in normaltissues. LyP-1 has been shown to deliver imaging agents intoatherosclerotic plaques and carotid inflammatory lesions, allowingenhanced imaging of the lesions (Fogal et al., 2008; Hamzah et al.,2011). Recent data also show that LyP-1 possesses a biological activitybeyond the homing and carrier functions; prolonged treatment ofatherosclerotic mice with this peptide has a plaque-reducing effect.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments, the presently disclosed subject matter providesisolated peptides or peptidomimetics. In some embodiments, the presentlydisclosed isolated peptides or peptidomimetics comprise the amino acidsequence KRGARST (SEQ ID NO: 1) or a peptidomimetic thereof, the aminoacid sequence AKRGARSTA (SEQ ID NO: 2) or a peptidomimetic thereof, orthe amino acid sequence CKRGARSTC (SEQ ID NO: 3) or a peptidomimeticthereof. In some embodiments, the presently disclosed peptide orpeptidomimetic is a peptide, optionally a linear peptide. In someembodiments, the isolated peptide or peptidomimetic is conformationallyconstrained. In some embodiments, the isolated peptide or peptidomimeticis cyclic. The presently disclosed isolated peptides or peptidomimeticshave a length of in some embodiments less than 100 residues, in someembodiments less than 50 residues, in some embodiments less than 20residues, and in some embodiments less than 15 residues.

The presently disclosed subject matter also provides in some embodimentsconjugates comprising one or more moieties linked to one or more homingmolecules that selectively home to tumor lymphatic vasculature, whereinthe one or more homing molecules comprise the presently disclosedisolated peptides and/or peptidomimetics. In some embodiments, theconjugates further comprise one or more additional homing molecules,which in some embodiments comprise one or more antibodies orantigen-binding fragments thereof. In some embodiments, at least one ofthe one or more homing molecules is a peptide, optionally a peptidecomprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The peptide orpeptidomimetic portions of the conjugates have in some embodiments alength of at most 200 residues, in some embodiments a length of at most100 residues, in some embodiments a length of at most 50 residues, insome embodiments a length of at most 20 residues, in some embodiments alength of at most 15 residues, and in some embodiments a length of atmost 10 residues. In some embodiments, at least one of the homingmolecules is conformationally constrained or cyclic. In someembodiments, at least one of the homing molecules is linear. In someembodiments, at least one of the homing molecules comprises the aminoacid sequence KRGARST (SEQ ID NO: 1), AKRGARSTA (SEQ ID NO: 2),CKRGARSTC (SEQ ID NO: 3), or a conservative variant or peptidomimeticthereof. In some embodiments, at least one of the homing moleculesconsists of the amino acid sequence KRGARST (SEQ ID NO: 1), AKRGARSTA(SEQ ID NO: 2), CKRGARSTC (SEQ ID NO: 3), or a conservative variant orpeptidomimetic thereof.

In some embodiments of the presently disclosed conjugates, the moietycomprises a therapeutic agent, which in some embodiments can be a cancerchemotherapeutic agent, a cytotoxic agent, and/or ananti-lymphangiogenic agent. In some embodiments, the moiety is adetectable agent. In some embodiments, the moiety is a phage.

The conjugates can also comprise in some embodiments at least two, ten,20, 25, 50, 100, 500, or 1000 homing molecules that each selectivelyhomes to tumor lymphatic vasculature, which in some embodiments eachindependently comprise and/or consist of the amino acid sequence KRGARST(SEQ ID NO: 1), AKRGARSTA (SEQ ID NO: 2), CKRGARSTC (SEQ ID NO: 3), or aconservative variant or peptidomimetic thereof.

The presently disclosed subject matter also provides in some embodimentsmethods for directing a moiety to tumor lymphatic vasculature in asubject. In some embodiments, the presently disclosed methods compriseadministering to the subject a conjugate that comprises a moiety linkedto a homing molecule that selectively homes to tumor lymphaticvasculature, wherein the homing molecule comprises an isolated peptideand/or peptidomimetic as disclosed herein, thereby directing the moietyto tumor lymphatic vasculature.

The presently disclosed subject matter also provides in some embodimentsmethods for imaging tumor lymphatic vasculature in a subject. In someembodiments, the methods comprise administering to the subject aconjugate comprising a detectable agent linked to a homing molecule thatselectively homes to tumor lymphatic vasculature, wherein the homingmolecule comprises a isolated peptide or peptidomimetic as disclosedherein; and detecting the conjugate, thereby imaging the tumor lymphaticvasculature.

The presently disclosed subject matter also provides in some embodimentsmethods for reducing the number of tumor lymphatic vessels in a subject.In some embodiments, the method comprise administering to the subject aconjugate that comprises a moiety linked to a homing molecule thatselectively homes to tumor lymphatic vasculature, wherein the homingmolecule comprises an isolated peptide or peptidomimetic as disclosedherein, thereby reducing the number of tumor lymphatic vessels in thesubject

The presently disclosed subject matter also provides in some embodimentsmethods for reducing or inhibiting tumor metastasis in a subject. Insome embodiments, the presently disclosed methods comprise administeringto the subject a conjugate that comprises a moiety linked to a homingmolecule that selectively homes to tumor lymphatic vasculature, whereinthe homing molecule comprises an isolated peptide or peptidomimetic asdisclosed herein, thereby reducing or inhibiting tumor metastasis in thesubject.

The presently disclosed subject matter also provides in some embodimentsmethods for treating cancer in a subject. In some embodiments, thepresently disclosed methods comprise administering to the subject aconjugate which comprises a moiety linked to a homing molecule thatselectively homes to tumor lymphatic vasculature, wherein the homingmolecule comprises an isolated peptide or peptidomimetic as disclosedherein, and further wherein the conjugate has an anti-cancer biologicalactivity in the tumor lymphatic vasculature of the subject.

The presently disclosed subject matter also provides in some embodimentscompositions comprising a surface molecule, one or more homingmolecules, and a plurality of membrane perturbing molecules, wherein thehoming molecule selectively homes to tumor vasculature. In someembodiments, one or more of the homing molecules comprise the amino acidsequence KRGARST (SEQ ID NO: 1) or a conservative derivative thereof,the amino acid sequence AKRGARSTA (SEQ ID NO: 2) or a conservativederivative thereof, the amino acid sequence CKRGARSTC (SEQ ID NO: 3) ora conservative derivative thereof, or any combination thereof. In someembodiments, one or more of the membrane perturbing molecules comprisethe amino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42) or a conservativevariant thereof, (KLAKLAK)₂ (SEQ ID NO: 42) or a conservative variantthereof, (KLAKKLA)₂ (SEQ ID NO: 43) or a conservative variant thereof,(KAAKKAA)₂ (SEQ ID NO: 44) or a conservative variant thereof, or(KLGKKLG)₃ (SEQ ID NO: 45) or a conservative variant thereof, or anycombination thereof. In some embodiments, one or more of the membraneperturbing molecules are conjugated to one or more of the homingmolecules. In some embodiments, one or more of the conjugated membraneperturbing molecules and homing molecules are covalently coupled. Insome embodiments, one or more of the covalently coupled membraneperturbing molecules and homing molecules comprise fusion peptides. Insome embodiments, the homing molecules are conjugated with the surfacemolecule. In some embodiments, one or more of the conjugated homingmolecules are indirectly conjugated to the surface molecule. In someembodiments, one or more of the conjugated homing molecules are directlyconjugated to the surface molecule. In some embodiments, one or more ofthe homing molecules are covalently coupled to the surface molecule. Insome embodiments, one or more of the covalently coupled homing moleculesare indirectly covalently coupled to the surface molecule. In someembodiments, one or more of the covalently coupled homing molecules aredirectly covalently coupled to the surface molecule. In someembodiments, the membrane perturbing molecules are conjugated with thesurface molecule. In some embodiments, one or more of the conjugatedmembrane perturbing molecules are indirectly conjugated to the surfacemolecule. In some embodiments, one or more of the conjugated membraneperturbing molecules are directly conjugated to the surface molecule. Insome embodiments, one or more of the membrane perturbing molecules arecovalently coupled to the surface molecule. In some embodiments, one ormore of the covalently coupled membrane perturbing molecules areindirectly covalently coupled to the surface molecule. In someembodiments, one or more of the covalently coupled membrane perturbingmolecules are directly covalently coupled to the surface molecule. Insome embodiments, one or more of the conjugated homing molecules areindirectly conjugated to the surface molecule via a linker, one or moreof the conjugated membrane perturbing molecules are indirectlyconjugated to the surface molecule via a linker, or both.

In some embodiments, the presently disclosed compositions furthercomprise a plurality of linkers. In some embodiments, at least one ofthe linkers comprises polyethylene glycol.

In some embodiments, the presently disclosed compositions furthercomprise one or more internalization elements. In some embodiments, oneor more of the homing molecules comprise one or more of theinternalization elements. In some embodiments, one or more of themembrane perturbing molecules comprise one or more of theinternalization elements. In some embodiments, the surface moleculecomprises one or more of the internalization elements not comprised ineither the homing molecules or the membrane perturbing molecules.

In some embodiments, the presently disclosed compositions furthercomprise one or more tissue penetration elements. In some embodiments,one or more of the tissue penetration elements are comprised in aninternalization element. In some embodiments, the tissue penetrationelement is a CendR element.

In some embodiments, the presently disclosed compositions bind insidetumor blood vessels.

In some embodiments, the presently disclosed compositions areinternalized in cells.

In some embodiments, the presently disclosed composition penetratestissue.

In some embodiments, the presently disclosed compositions reduce tumorgrowth.

In some embodiments of the presently disclosed compositions, the surfacemolecule comprises an nanoparticle, a nanoworm, an iron oxide nanoworm,an iron oxide nanoparticle, an albumin nanoparticle, a liposome, amicelle, a phospholipid, a polymer, a microparticle, and/or afluorocarbon microbubble.

In some embodiments, the presently disclosed compositions comprise atleast 100, 1000, or 10,000 homing molecules, and/or comprise at least100, 1000, or 10,000 membrane perturbing molecules.

In some embodiments of the presently disclosed compositions, one or moreof the homing molecules are modified homing molecules, which in someembodiments are methylated homing molecules, optionally wherein one ormore of the methylated homing molecules comprise a methylated amino acidsegment, further optionally wherein the amino acid sequence is N- orC-methylated in at least one position; and/or one or more of themembrane perturbing molecules are modified membrane perturbingmolecules, which in some embodiments are methylated membrane perturbingmolecules, optionally wherein one or more of the methylated membraneperturbing molecules comprise a methylated amino acid segment, furtheroptionally wherein the amino acid sequence is N- or C-methylated in atleast one position.

In some embodiments of the presently disclosed compositions, thecompositions further comprise one or more moieties, which in someembodiments are independently selected from the group consisting of atherapeutic agent, an anti-angiogenic agent, a pro-angiogenic agent, acancer chemotherapeutic agent, a cytotoxic agent, an anti-inflammatoryagent, an anti-arthritic agent, a polypeptide, a nucleic acid molecule,a small molecule, an image contrast agent, a fluorophore, fluorescein,rhodamine, a radionuclide, indium-111, technetium-99, carbon-11, andcarbon-13. In some embodiments, the therapeutic agent is selected fromthe group consisting of iRGD, Abraxane, paclitaxel, and taxol. In someembodiments, at least one of the moieties is a detectable agent,optionally FAM.

In some embodiments, the presently disclosed subject matter provides insome embodiments a method comprising administering to a subjectcomposition of the presently disclosed subject matter, wherein thecomposition selectively homes to tumor vasculature in the subject,wherein the composition is internalized into cells at the site of thetumor vasculature, optionally wherein the composition has a therapeuticeffect, which in some embodiments comprises a slowing in the increase ofor a reduction of tumor burden and in some embodiments comprises aslowing of the increase of or reduction of tumor size. In someembodiments, the subject has one or more sites to be targeted, whereinthe composition homes to one or more of the sites to be targeted. Insome embodiments, the subject has a tumor, wherein the composition has atherapeutic effect on the tumor. In some embodiments, the compositionpenetrates tissue, and in some embodiments the composition penetratestumor tissue.

The presently disclosed subject matter also provides in some embodimentsmethods for treating a disease or disorder associated with a gC1q/p32receptor biological activity. In some embodiments, the methods compriseidentifying a subject having a disease or disorder associated with agC1q/p32 receptor biological activity; and administering to the subjecta composition comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Insome embodiments, the subject has cancer. In some embodiments, thecomposition further comprises a moiety, optionally a therapeutic moiety,a diagnostic agent, and/or a nanoparticle. In some embodiments, thetherapeutic moiety targets a DNA-associated process.

The presently disclosed subject matter also provides in some embodimentsmethods for detecting the presence of gC1q/p32 receptor. In someembodiments, the methods comprise bringing into contact a cell and a TT1Peptide composition, wherein the TT1 Peptide composition comprises amoiety linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3; and detecting interaction between gC1q/p32 receptor andthe TT1 Peptide composition, thereby detecting the presence of gC1q/p32receptor. In some embodiments, the moiety is a detectable agent, apolypeptide, a nucleic acid molecule, or a small molecule. In someembodiments, the TT1 Peptide composition comprises a virus, which is insome embodiments a phage.

The presently disclosed subject matter also provides in some embodimentsmethods for detecting interactions between a gC1q/p32 receptor and a TT1Peptide composition. In some embodiments, the TT1 Peptide compositioncomprises a moiety linked to a composition comprising SEQ ID NO: 1, SEQID NO: 2, or SEQ ID NO: 3. In some embodiments, the methods compriseselecting a cell for its potential to comprise a gC1q/p32 receptor;bringing into contact the TT1 Peptide composition and the cell; anddetecting interaction between the gC1q/p32 receptor and the TT1 Peptidecomposition.

The presently disclosed subject matter also provides in some embodimentsmethods for delivering TT1 Peptide compositions to gC1q/p32 receptors.In some embodiments, the TT1 Peptide compositions comprise one or moremoieties linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2,or SEQ ID NO: 3. In some embodiments, the methods comprise bringing intocontact the TT1 Peptide composition and a cell, thereby delivering theTT1 Peptide composition to the gC1q/p32 receptor. In some embodiments,the cell is in a subject, wherein the cell is selected for its potentialto comprise a gC1q/p32 receptor by detecting the presence of gC1q/p32receptor on another cell of the subject.

The presently disclosed subject matter also provides in some embodimentsmethods for delivering TT1 Peptide compositions to gC1q/p32 receptors.In some embodiments, the TT1 Peptide compositions comprise one or moremoieties linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2,or SEQ ID NO: 3. In some embodiments; the methods comprise selecting acell for its potential to comprise a gC1q/p32 receptor; and bringinginto contact the TT1 Peptide composition and the cell, therebydelivering the TT1 Peptide composition to the gC1q/p32 receptor.

The presently disclosed subject matter also provides methods forassessing gC1q/p32 receptor levels in cells of a subject. In someembodiments, the methods comprise bringing into contact a cell of thesubject and a TT1 Peptide composition comprising a detectable agentlinked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO: 3; and detecting the level of the TT1 Peptide compositioninteracting with gC1q/p32 receptor, thereby assessing gC1q/p32 receptorlevel in the cell. In some embodiments, the level of gC1q/p32 receptorin the subject is compared to a previous measurement in the same subjectand/or is compared to a control level or standard level.

The presently disclosed subject matter also provides in some embodimentsmethods for identifying subjects having a disease or disorder associatedwith a gC1q/p32 receptor biological activity. In some embodiments, themethods comprise bringing into contact a cell of the subject and a TT1Peptide composition, wherein the TT1 Peptide composition comprises amoiety linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3; and detecting interaction between gC1q/p32 receptor andthe TT1 Peptide composition, thereby detecting the presence or level ofgC1q/p32, wherein the presence or level of gC1q/p32 receptor identifiesthe subject as having a disease or disorder associated with a gC1q/p32receptor biological activity. In some embodiments, the disease ordisorder is cancer or inflammation. In some embodiments, the cell is acancerous cell.

The presently disclosed subject matter also provides in some embodimentsmethods for screening for a compound that interacts with a gC1q/p32receptor. In some embodiments, the methods comprise bringing intocontact a test compound, a TT1 Peptide composition, and a gC1q/p32receptor, wherein the TT1 Peptide composition comprises SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3; and detecting unbound TT1 Peptidecomposition, wherein a given amount of unbound TT1 Peptide compositionindicates a compound that interacts with gC1q/p32 receptor. In someembodiments, the TT1 Peptide composition further comprises a moietylinked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO: 3. In some embodiments, the moiety further comprises a detectableagent.

The presently disclosed subject matter also provides in some embodimentsmethods for treating a disease or disorder associated with a gC1q/p32receptor biological activity. In some embodiments, the methods compriseidentifying a subject having a disease or disorder associated with agC1q/p32 receptor biological activity; and administering to the subjecta composition that interacts with the gC1q/p32 receptor, wherein thecomposition comprises a TT1 Peptide comprising SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3, thereby treating a disease or disorder associatedwith a gC1q/p32 receptor biological activity.

With respect to any of the compositions, conjugates, and methods of thepresently disclosed subject matter, in some embodiments the homingmolecule is linear or cyclic, and/or is a peptide or peptidomimetic. Insome embodiments, the homing molecule comprises the amino acid sequenceKRGARST (SEQ ID NO: 1) or a conservative variant or peptidomimeticthereof, the amino acid sequence AKRGARSTA (SEQ ID NO: 2) or aconservative variant or peptidomimetic thereof, and/or the amino acidsequence CKRGARSTC (SEQ ID NO: 3) or a conservative variant orpeptidomimetic thereof. In some embodiments, the moiety is, comprises,consists essentially of, or consists of a therapeutic agent, a cancerchemotherapeutic agent, a cytotoxic agent, an anti-lymphangiogenicagent, a detectable agent, a phage, a polypeptide, a nucleic acidmolecule, a small molecule, a fluorophore, fluorescein, rhodamine, aradionuclide, indium-111, technetium-99, carbon-11, carbon-13, or anycombination thereof. In some embodiments, the therapeutic moiety isselected from the group consisting of a cytotoxic agent, an alkylatingagent, an anti-tumor antibiotic, a sequence-selective agent, ananti-angiogenic agent, cyclophosphamide, melphalan, mitomycin C,bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone, SN-38,Et-743, actinomycin D, bleomycin, geldanamycin, chlorambucil,methotrexate, and TLK286. In some embodiments, the detectable agent is,comprises, consists essentially of, or consists of a radionuclide, whichin some embodiments is selected from the group consisting of indium-111,technetium-99, carbon-11, and carbon-13. In some embodiments, the one ormore of the membrane perturbing molecules comprise, consist essentiallyof, or consist of the amino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO:42), (KLAKLAK)₂ (SEQ ID NO: 42), (KLAKKLA)₂ (SEQ ID NO: 43), (KAAKKAA)₂(SEQ ID NO: 44), and/or (KLGKKLG)₃ (SEQ ID NO: 45), or any combinationthereof. In some embodiments, one or more of the homing moleculescomprise, consist essentially of, and/or consist of the amino acidsequence KRGARST (SEQ ID NO: 1), AKRGARSTA (SEQ ID NO: 2), and/orCKRGARSTC (SEQ ID NO: 3), wherein one or more of the membrane perturbingmolecules comprise, consist essentially of, and/or consist of the aminoacid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42), wherein one or more of theconjugated homing molecules are indirectly conjugated to the surfacemolecule via a linker, and wherein one or more of the conjugatedmembrane perturbing molecules are indirectly conjugated to the surfacemolecule via a linker, optionally a polyethylene glycol (PEG) linker.

Thus, it is an object of the presently disclosed subject matter toprovide compositions and methods for delivering active agents tosubjects.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingdrawings as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one Figure executed incolor. Copies of this patent or patent application publication withcolor Figures will be provided by the Office upon request and payment ofthe necessary fee.

The accompanying drawings, which are incorporated in and constitute apart of this disclosure, illustrate several embodiments of the disclosedmethods and compositions, and together with the description, serve toexplain the principles of the disclosed methods and compositions.

FIGS. 1A and 1B depict the results of purification and oligomerizationanalysis of the p32 protein. FIG. 1A is a picture of an analyticalSDS-PAGE gel of 6×-His-Tagged p32 expressed in Rosetta-gami-2 cells(Novagen). FIG. 1B is a plot of a sedimentation velocity assay.

FIGS. 2A and 2B depict the results of in vitro biopanning of T7bacteriophage libraries on immobilized p32 protein. FIG. 2A is a bargraph showing the results of two peptide libraries (CX7C and X7;C=cysteine; X=any amino acid) that were used for in vitro biopanning onimmFigure lobilized p32. Binding is expressed as fold over control phagedisplaying polyglycine heptapeptide (G7). FIG. 2B presents WebLogo(Schneider & Stephens, 1990; Crooks et al., 2004) consensus peptidemotifs (XRGXRS; SEQ ID NO: 197 and CXRGXRXXC; SEQ ID NO: 198) andrepresentative peptide sequences recovered after two rounds of ex vivoselections. For the linear library, the KLAALE (SEQ ID NO: 11) elementwas encoded by the phage genomic DNA and was thus not a part of therandom library. The data are representative of four (4) independentscreens and binding experiments.

FIG. 3 is a bar graph of binding of individual RGXRS (SEQ ID NO:4)-displaying phage clones to immobilized p32 and NRP-1 b1b2 domain. Invitro binding experiments were performed using phage clones displayingp32-selected RGRS (SEQ ID NO: 13)-containing peptides or phage clonesselected on NRP-1 b1b2 domain. Note the lack of cross-binding of thephage to different target proteins. LyP-1 (a known p32 binder; SEQ IDNO: 7) and RPARPAR (a prototypic CendR peptide that binds to NRP-1 b1b2domain; SEQ ID NO: 14) served as controls. The data are representativeof three (3) independent binding experiments. An exemplary P32-bindingCKRGARSTC peptide (TT1; SEQ ID NO: 3) is highlighted by a box. Peptidesfrom left to right in FIG. 3 are a control polyglycine heptapeptide(G7), RPARPAR (SEQ ID NO: 14), LYP1 (SEQ ID NO: 7), RMRRIDR (SEQ ID NO:190), SGVSRDR (SEQ ID NO: 191), SMNEVRKR (SEQ ID NO: (192), SRLGRMR (SEQID NO: 193), GRGGRSRKLAAALE (SEQ ID NO: 161), RKRGGRS (SEQ ID NO: 166),RRGGRSKLAAALE (SEQ ID NO: 170), CKRGARSTC (SEQ ID NO: 3), CARGARTKC (SEQID NO: 176), CKRGNRSVC (SEQ ID NO: 178), CKRGSRSSC (SEQ ID NO: 181),CQRGTRSRC (SEQ ID NO: 182), CTRGSRSKC (SEQ ID NO: 177), CVRGGRARC (SEQID NO: 183), and CARGKRSLC (SEQ ID NO: 184).

FIGS. 4A and 4B are plots showing a comparison of the binding of LyP-1(SEQ ID NO: 7; diamonds) and TT1 (SEQ ID NO: 3; squares) to p32 in afluorescence polarization assay. FIG. 2A is a plot showing that anexemplary TT1 peptide (CKRGARSTC; SEQ ID NO: 3) bound to p32 with ahigher affinity than LyP-1. FIG. 2B is a plot showing that the exemplaryTT1 peptide, the CendR motif of which is cryptic, showed only backgroundbinding, whereas the prototypic active CendR peptide RPARPAR (SEQ ID NO:14; triangles) avidly bound to NRP-1. NRP-1 was used as a controlprotein.

FIG. 5 is a plot showing that P32-TT1 (CKRGARSTC; SEQ ID NO: 3) andLyP-1 (SEQ ID NO: 7) peptides competed for binding to the P32 protein.The results were derived from a fluorescence polarization assay thatdemonstrated that binding of labeled TT1 (SEQ ID NO: 3) to P32 could beinhibited by unlabeled TT1 (SEQ ID NO: 3) and LyP-1 (SEQ ID NO: 7) butnot by RPARPAR peptide (SEQ ID NO: 14). Squares: p32-TT1; Diamonds:LyP-1; X: RPARPAR peptide (SEQ ID NO: 14).

FIGS. 6A and 6B are fluorescence micrographs showing internalization ofCKRGARSTC (TT1; SEQ ID NO: 3) peptide in breast tumor cells in vitro.100 μM FAM-labeled peptides were incubated with 4T1 (FIG. 6A) or MCF10(FIG. 6B) breast cancer cells for 1 hour at 37° C. in DMEM highglucose/10% serum medium. Cells were washed, fixed, stained withanti-FITC antibody, and MITOTRACKER® (Life Technologies) or phalloidinas indicated. Scale bars: 20 am.

FIGS. 7A and 7B are photographs depicting in vivo homing of an exemplaryTT1 peptide. 200 μg of FAM-labeled TT1 (SEQ ID NO: 3) peptide or controlpeptide in PBS was intravenously injected to mice bearing orthotopic 4T1(FIG. 7A) or MCF7 (FIG. 7B) breast tumors. The peptides were allowed tocirculate for 2 hours and organs were collected and photographed underwhite (top panels) or UV (bottom panels) light. Arrows point to thetumors of TT1 (SEQ ID NO: 3)-injected mice. Note robust signal (white inblack and white versions and green in color versions of the Figures)indicating homing of TT1 (SEQ ID NO: 3) peptide to the tumor in bothmodels. H: heart; Lu: lung; Li: liver; Ki: kidney; Sp: spleen; T: tumor.

FIG. 8 is a series of fluorescence micrographs showing TT1 (SEQ ID NO:3) peptide homed to and penetrated into plaque tissue as well as orbetter than LyP-1 (SEQ ID NO: 7). ApoE null mice that had been kept on ahigh-fat diet were intravenously injected with 100 mg of the FAM labeledpeptides. One hour later, the mice were perfused through the heart underanesthesia, and tissues were collected. The panels show accumulation ofFAM labeled peptides visualized with UV excitation. The center two andright two panels show atherosclerotic aortas that were sectioned at theaortic root level and examined by confocal microscopy. LyP-1 (SEQ ID NO:7) signal (center two panels) and a stronger TT1 (SEQ ID NO: 3) signal(right two panels) were seen inside plaques, whereas a control peptide(left two panels) did not accumulate in the plaques.

FIG. 9 is a bar graph showing proteolytic switching of bindingspecificity of an exemplary TT1 peptide (SEQ ID NO: 3). T7 phagedisplaying an exemplary TT1 peptide (SEQ ID NO: 3) were exposed toincreasing concentrations of crystalline trypsin and incubated for 30minutes at 37° C. After treatment, phage were incubated with magneticbeads coated with either p32 (decending hatching left to right) or NRP-1b1b2 (ascending hatching left to right) protein, followed by washes toremove unbound phage and quantitation of specifically bound phage.

FIG. 10 is a bar graph showing that the p32 binding of the RGXR(S/T)motif (SEQ ID NOs: 4 and 5) in the exemplary TT1 peptide (SEQ ID NO: 3)was not dependent on the cyclic structure and compatible with flankingsequences. Phage clones displaying the indicated peptides were incubatedin p32-coated magnetic microbeads. After washing to remove non-boundphage, specifically bound phage were quantified. Note that a TT1 variant(SEQ ID NO: 2) with cysteines replaced by alanines retained the bindingactivity and that the binding was not reduced by flanking (T)GGSGlinkers (SEQ ID NOs: 15 and 16).

FIG. 11 depicts the design of an exemplary theranostic nanosystem. Achimeric peptide combining a tumor-homing peptide (a TT1 Peptide in thisexample; SEQ ID NO: 3) with a pro-apoptotic peptide (KLAKLAKKLAKLAK; SEQID NO: 42) was covalently coupled to iron oxide nanoworms (NWs; length80-100 nm, width 30 nm; Park et al., 2008). An extra cysteine was addedto the N-terminus of the cyclic LyP-1 (SEQ ID NO: 199) nonapeptide andused for coupling NWs. The drug peptide and the fluorophore wereattached to the free N-terminus of the same cysteine residue.

FIG. 12 is a fluorescent micrograph showing tumor homing of linearTT1-NWs. Tumor-bearing mice were intravenously injected withFAM-LinTT1-coated NWs (7.5 mg iron/kg) and allowed to circulate for 5hours. The mice were perfused through the heart with PBS, and tumors andorgans were collected and processed for fluorescence microscopy. Theleft panel is a representative confocal microscopy image from tumors inmice. The right panel is a merged image showing the presence of NWs(white in black and white versions and green in color versions of FIG.12), CD31 (light gray in black and white versions and red in colorversions of FIG. 12); and nuclei (dark gray in black and white versionsand blue in color versions of FIG. 12). Scale bars: 100 am. The imagesshow striking NW accumulation outside tumor blood vessels.

FIG. 13 is a bar graph showing quantification of the homing of variouspeptide-coated NWs. NWs were coated with the indicated peptides (CGKRK(SEQ ID NO: 6); LyP-1 (SEQ ID NO: 7); a cyclic TT1 peptide (SEQ ID NO:3); or a linear TT1 peptide (SEQ ID NO: 2)) and intravenously injectedinto mice (n=3 per peptide) bearing orthotropic breast tumors. The dosewas 7.5 mg/kg body weight of NW iron. The NWs were allowed to circulatefor 5 hours, after which the mice were perfused through the heart withPBS and tumors and organs were collected. Quantification of fluorescencewas done with ImageJ software (available from the website of the UnitedStates National Institutes of Health). In addition to the tumors, NWswere detected in the liver and spleen, which non-specifically capturenanoparticles regardless of their peptide coating. Other organs did notcontain significant numbers of NWs, confirming the specificity of thetumor homing.

FIG. 14 is a series of fluorescent micrographs showing tumor homing ofLinTT1-_(D)[KLAKLAK]₂-NWs (SEQ ID NO: 10). Confocal images show someco-localization of NW fluorescence (middle panel; white in black andwhite versions and green in color versions of FIG. 14) with bloodvessels (CD31; left panel; white in black and white versions and red incolor versions of FIG. 14), but most of the NWs are outside the bloodvessels in the extravascular tumor tissue. The right panel is a mergedimage (NWs: white in black and white versions and green in colorversions of FIG. 14; CD31: light gray in black and white versions andred in color versions of FIG. 14; nuclei: dark gray in black and whiteversions and blue in color versions of FIG. 14). Scale bars are 100 m.

FIG. 15 is a bar graph showing tumor accumulation of NWs coated withchimeric peptides consisting of a homing peptide and pro-apoptoticpeptide. Quantification of NW fluorescence was done with ImageJsoftware. FAM Intensity as % Area is plotted on a scale of 0-10 for thefollowing peptide-conjugated NWs: 1—CGKRK-_(D)[KLAKLAK]₂-NWs (SEQ ID NO:8); 2—LyP-1-_(D)[KLAKLAK]₂-NWs (SEQ ID NO: 9);3—LinTT1-_(D)[KLAKLAK]₂-NWs (SEQ ID NO: 10).

FIG. 16 is a graph of tumor volume of orthotopic MCF10-CA1a breastcancer cell tumors at days 0-20 after treatment with vehicle only (PBS;inverted triangles), linear TT1 Peptide (SEQ ID NO: 2) conjugatednanoworms without the drug peptide ((TT1-NW; triangles),CGKRK-_(D)(KLAKLAK)₂—NWs (squares; SEQ ID NO: 8), or linearTT1-_(D)(KLAKLAK)₂—NWs (circles; SEQ ID NO: 10). Error bars relate tostandard deviation as calculated by ANOVA.

FIG. 17 is a graph depicting the results of tumor treatment withLinTT1-_(D)[KLAKLAK]₂ (SEQ ID NO: 10)-micelles in a MCF10CA1a breastcancer model. Black circles: PBS control; white circles:LinTT1-_(D)[KLAKLAK]₂ (SEQ ID NO: 10)-micelles.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-3 are the amino acid sequences of exemplary TT1 Peptides.

SEQ ID NOs: 4 and 5 are the amino acid sequences of a pentatpeptidemotif that was highly represented in phage-displayed peptides that boundto the p32 protein.

SEQ ID NO: 6 is the amino acid sequence of an exemplary p32-bindingpeptide.

SEQ ID NO: 7 is the amino acid sequence of exemplary p32-binding peptideLyP-1.

SEQ ID NO: 8 is the amino acid sequence of an exemplary p32-bindingpeptide conjugated to a membrane-perturbing sequence.

SEQ ID NO: 9 is the amino acid sequence of an exemplary Lyp-1Peptide-based p32-binding peptides conjugated to a membrane-perturbingsequence.

SEQ ID NO: 10 is the amino acid sequence of an exemplary TT1Peptide-based p32-binding peptides conjugated to a membrane-perturbingsequence.

SEQ ID NO: 11 is the amino acid sequence of an element was encoded bythe phage genomic DNA and was thus not a part of the random linearlibrary.

SEQ ID NO: 12 is the amino acid sequence of a peptapeptide core presentin certain exemplary TT1 Peptides.

SEQ ID NO: 13 is the amino acid sequence present in phage clonesemployed in certain in vitro p32 protein binding experiments.

SEQ ID NO: 14 is the amino acid sequence of a prototypic CendR peptidethat binds to NRP-1 b1b2 domain.

SEQ ID NOs: 15 and 16 are the amino acid sequences of exemplary linkers.

SEQ ID NOs: 17 and 42-45 are the amino acid sequences of exemplarymembrane-perturbing peptides.

SEQ ID NOs: 18-41 are the nucleotide and amino acid sequences ofexemplary p32 gene products. Within SEQ ID NOs: 18-41, the even numberedsequences are exemplary nucleotide sequences and the odd-numberedsequences are the amino acid sequences encoded by the immediatelypreceding SEQ ID NO.

SEQ ID NOs: 42-159 are the amino acid sequences of exemplary homingpeptides.

SEQ ID NOs: 160-174 are the amino acid sequences from peptides recoveredfrom the X7 library.

SEQ ID NOs: 175-189 are the amino acid sequences from peptides recoveredfrom the CX7C library.

SEQ ID NOs: 190-193 are the amino acid sequences of peptides recoveredfrom phage that were selected against the NRP-1 b1 b2 domain.

SEQ ID NOs: 194-196 are the amino acid sequences of peptides recoveredfrom phage that were selected against the p32 protein.

SEQ ID NOs: 197 and 198 are the WebLogo consensus sequences for peptidesisolated from the X7 and CX7C libraries, respectively.

SEQ ID NO: 199 is the sequence of an LyP-1 peptides with an extracysteine added to its N-terminus.

DETAILED DESCRIPTION

The disclosed method and compositions can be understood more readily byreference to the following detailed description of exemplaryembodiments, the EXAMPLES included therein, and to the Figures and theirprevious and following descriptions.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

I. Definitions

As used in the disclosure and the appended claims, the singular forms“a,” “an”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

As used in the claims, the term “comprising”, which is synonymous with“including”, “containing”, and “characterized by”, is inclusive oropen-ended and does not exclude additional, unrecited elements and/ormethod steps. “Comprising” is a term of art that indicates that thenamed elements and/or steps are present, but that other elements and/orsteps can be added and still fall within the scope of the relevantsubject matter.

As used herein, the phrase “consisting of” excludes any element, step,and/or ingredient not specifically recited. For example, when the phrase“consists of” appears in a clause of the body of a claim, rather thanimmediately following the preamble, it limits only the element set forthin that clause; other elements are not excluded from the claim as awhole.

As used herein, the phrase “consisting essentially of” limits the scopeof the related disclosure or claim to the specified materials and/orsteps, plus those that do not materially affect the basic and novelcharacteristic(s) of the disclosed and/or claimed subject matter. Forexample, the peptides of the presently disclosed subject matter in someembodiments can “consist essentially of” a recited amino acid sequence,which indicates that the peptide can include one or more (e.g., 1, 2, 3,4, 5, 6, or more) N-terminal and/or C-terminal amino acids the presenceof which does not materially affect the desired biological activity ofthe peptide. In the context of the peptides disclosed herein, in someembodiments a peptide that “consists essentially of” a particularlyenumerated amino acid sequence includes only that amino acid sequencebut can include one or more additional amino acids at the N- and/orC-terminus (e.g., 1, 2, 3, 4, 5, or 6 amino acids at one or bothtermini) provided that the presence of the additional amino acids doesnot significantly (e.g., by no more than +5%, +10%, +15%, +20%, +25%,+30%, +35%, +35%, +40%, +45%, or +50^(%)) increase or decrease thebiological activity desired for the peptide as compared to the samebiological activity of the peptide lacking the additional N- and/orC-terminal amino acids.

With respect to the terms “comprising”, “consisting essentially of”, and“consisting of”, where one of these three terms is used herein, thepresently disclosed subject matter can include the use of either of theother two terms. For example, the presently disclosed subject matterrelates in some embodiments to compositions comprising the amino acidsequence KRGARST (SEQ ID NO: 1). It is understood that the presentlydisclosed subject matter thus also encompasses peptides that in someembodiments consist essentially of the amino acid sequence KRGARST (SEQID NO: 1); as well as peptides that in some embodiments consist of theamino acid sequence KRGARST (SEQ ID NO: 1). Similarly, it is alsounderstood that the methods of the presently disclosed subject matter insome embodiments comprise the steps that are disclosed herein and/orthat are recited in the claims, that they in some embodiments consistessentially of the steps that are disclosed herein and/or that arerecited in the claims, and that they in some embodiments consist of thesteps that are disclosed herein and/or that are recited in the claim.

For all terms defined herein below, grammatical variants are alsoencompassed by the definitions provided. For example, the term“modulate” is defined herein, and the definition provided is understoodto apply mutatis mutandis to the terms “modulating”, “modulated”,“modulates”, “modulator”, etc.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data are provided in a number of differentformats, and that these data represent endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point “15” aredisclosed, it is understood that greater than, greater than or equal to,less than, less than or equal to, and equal to 10 and 15 are considereddisclosed as well as between 10 and 15. It is also understood that eachunit between two particular units are also disclosed. For example, if 10and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.Similarly, when the context allows for data values to be expressed withone or more decimal places, each and every individual value between twodata values that employ the relevant number of decimal places is alsodisclosed. By way of example and not limitation, if 10 and 12 aredisclosed, it is understood that 10.1, 10.2, 10.3, 10.4, 10.5, 10.6,10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,and 11.9 are also disclosed.

In this disclosure and in the claims that follow, reference will be madeto a number of terms which shall be defined to have the followingmeanings:

The terms “optional” and “optionally” mean that the subsequentlydescribed event or circumstance might or might not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. Similarly, optional elements are eitherpresent or absent in various embodiments of the presently disclosedsubject matter, and it is understood that a claim that recites one ormore optional elements encompasses the situation where one, some, or allof the optional elements are either present or absent in any combinationor subcombination.

The term “activity” as used herein refers to a measurable result of theinteraction of molecules. Some exemplary methods of measuring theseactivities are provided herein.

The term “modulate” as used herein refers to the ability of a compoundor composition to change an activity (in some embodiments, a biologicalactivity) in some measurable way as compared to an appropriate control.As a result of the presence of compounds in the assays, activities canincrease or decrease as compared to controls in the absence of thesecompounds or compositions. An increase in an activity can be at least10%, in some embodiments at least 20%, in some embodiments at least 25%,in some embodiments at least 50%, in some embodiments at least 60%, insome embodiments at least 70%, in some embodiments at least 75%, in someembodiments at least 80%, in some embodiments at least 85%, in someembodiments at least 90%, in some embodiments at least 95%, in someembodiments at least 100%, and in some embodiments greater than 100%compared to the level of activity in the absence of the compound orcomposition. Similarly, a decrease in an activity can be at least 10%,in some embodiments at least 20%, in some embodiments at least 25%, insome embodiments at least 50%, in some embodiments at least 60%, in someembodiments at least 70%, in some embodiments at least 75%, in someembodiments at least 80%, in some embodiments at least 85%, in someembodiments at least 90%, in some embodiments at least 95%, and in someembodiments at least 100% compared to the level of activity in theabsence of the compound or composition. A modulator (in some embodimentsa compound) that increases an activity is in some embodiments referredto as an “agonist”, and one that decreases, or prevents an activity isin some embodiments referred to as an “antagonist.”

As used herein, the term “inhibit” refers to reducing or decreasing anactivity or expression. This can be a complete inhibition of activity orexpression, or a partial inhibition. In some embodiments, inhibition canbe compared to a control or to a standard level. Inhibition can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% as compared to a given control or standardlevel of activity or expression.

The term “monitoring” as used herein refers to any method by which anactivity, including but not limited to a biological activity, can bemeasured.

The term “providing” as used herein in the context of reagents refers toany method of adding a compound or molecule to something known in theart. EXAMPLES of providing can include the use of pipettes, pipettors,syringes, needles, tubing, guns, etc. Providing can be manual orautomated. In some embodiments, it can include transfection by anymethodology or any other method for providing nucleic acids to dishes,cells, tissue, and/or cell-free systems, and can relate to in vitro orin vivo manipulations.

The term “providing” as used herein in the context of a subject orpatient refers to making the subject or patient, and/or a biologicalsample isolated therefrom, available for treatment and/or diagnosis.

As used herein, the term “preventing” refers to administering a compoundprior to the onset of clinical symptoms of a disease or conditions so asto prevent a physical manifestation of aberrations associated with thedisease or condition.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms in order to ameliorate at least onesuch clinical symptom or any complication resulting therefrom.

The phrase “in need of treatment” as used herein refers to a judgmentmade by a caregiver (e.g., physician, nurse, nurse practitioner, orindividual in the case of humans; veterinarian in the case of animals,including non-human mammals) that a subject or patient requires or willbenefit from a given treatment. This judgment can be made based on avariety of factors that are in the realm of a care giver's expertise,but that can include the knowledge that the subject is ill, or will beill, as the result of a condition that is treatable by compounds of thepresently disclosed subject matter.

As used herein, the term “subject” includes, but is not limited to,animals, plants, bacteria, viruses, parasites, and any other organism orentity. The subject can in some embodiments be a vertebrate, in someembodiments a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, abird, a reptile, or an amphibian. The subject can in some embodiments bean invertebrate, in some embodiments an arthropod (e.g., an insects or acrustacean). The term does not denote a particular age or gender. Thus,adult and newborn subjects, as well as fetuses, whether male or female,are intended to be covered. The term “patient” refers to a subjectafflicted with a disease or disorder or potentially at risk fordeveloping a disease or disorder. The term “patient” includes in someembodiments humans and in some embodiments veterinary subjects.

The terms “higher, ““increases”, “elevates”, and “elevation” refer toincreases above basal levels, e.g., as compared to a control. The terms“low”, “lower”, “reduces”, and “reduction” refer to decreases belowbasal levels, e.g., as compared to a control.

Throughout the instant disclosure, various publications are referenced.The disclosures of these publications are hereby incorporated byreference in their entireties into this application in order to morefully describe the state of the art to which this pertains. Thereferences disclosed herein are also individually and specificallyincorporated by reference for the material contained in them that isdiscussed in the sentence in which the reference is relied upon.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, can vary. It is also to be understood that the terminology usedherein is for the purpose of describing exemplary embodiments only andis not intended to be limiting.

II. Compositions and Methods of Making the Same

Disclosed are exemplary components that can be used to prepare thedisclosed compositions as well as the compositions themselves to be usedwithin the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular peptide is disclosed and discussed and a numberof modifications that can be made to a number of molecules including thepeptide are discussed, specifically contemplated is each and everycombination and permutation of the peptides and the modifications thatare possible, unless specifically indicated to the contrary. Thus, if aclass of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited each isindividually and collectively contemplated meaning combinations, A-E,A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed.Likewise, any subset or combination of these is also disclosed. Thus,for example, the sub-group of A-E, B-F, and C-E would be considereddisclosed. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods.

II.A. TT1 and gC1qR/p32

Disclosed are compositions useful for delivering significant amounts ofcompounds of interest to targeted cells and tissues. The disclosedcompositions are useful, for example, to deliver to targeted cells andtissues an effective amount of compounds that are excessively toxic. Forexample, disclosed are compositions comprising a surface molecule, oneor more homing molecules, and a plurality of cargo molecules. The cargomolecules can be, for example, excessively toxic molecules. The cargomolecules can be, for example, membrane perturbing molecules. As anotherexample, disclosed are compositions comprising a surface molecule, oneor more homing molecules, and a plurality of membrane perturbingmolecules. As used herein, excessively toxic compounds are compoundsthat too toxic when administered to a subject in unconjugated forms inwhat would be a therapeutically effective amount but for the toxicity.

The homing molecules can home to targets of interest, such as cells andtissues of interest. For example, the homing molecules can home to tumorvasculature. The homing molecules can selectively home to targets ofinterest, such as cells and tissues of interest. For example, the homingmolecules can selectively home to tumor vasculature. The composition canhome to one or more of the sites to be targeted. The composition can beinternalized in cells. The composition can penetrate tissue. Thecomposition can be internalized into cells at the targeted site. Thecomposition can penetrate tissue at the targeted site. The compositioncan, for example be internalized into cancer cells. The composition can,for example, penetrate tumor tissue. The composition can, for example,bind inside tumor blood vessels.

Disclosed herein is the discovery that exemplary TT1 Family Peptides(including but not limited to KRGARST (SEQ ID NO: 1), AKRGARSTA (SEQ IDNO: 2), and CKRGARSTC (SEQ ID NO: 3); also referred to herein as “TT1”and/or “the TT1 Peptides”) selectively interacts with the gC1q receptor(gC1qR/p32, which has been described in the literature by one of thealternative terms gC1qR and p32, and is described herein as eithergC1qR, gC1q receptor, p32, or as gC1qR/p32, and which refers to theprotein known in the literature as gC1qR and as p32). gC1qR/p32 isassociated with tumor lymphatic vasculature, for example, the lymphaticvasculature of breast cancer tumors, squamous carcinomas, andosteosarcomas. gC1qR/p32 is also associated with inflammation (Waggoneret al., 2005, herein incorporated by reference in its entirety).Exemplary gC1qR nucleotide and amino acid sequences are presented hereinbelow in Table 1.

TABLE 1 Exemplary gC1qR Nucleotide and Amino Acid Sequences SpeciesNucleotide¹ Amino Acid¹ Homo sapiens NM_001212 NP_001203 (SEQ ID NO: 18)(SEQ ID NO: 19) Gorilla gorilla gorilla XM_004058391 XP_004058439 (SEQID NO: 20) (SEQ ID NO: 21) Pongo abelii XM_002826905 XP_002826951 (SEQID NO: 22) (SEQ ID NO: 23) Macaca fascicularis XM_005582637 XP_005582694(SEQ ID NO: 24) (SEQ ID NO: 25) Macaca mulatta XM_001100940 XP_001100940(SEQ ID NO: 26) (SEQ ID NO: 27) Callithrix jacchus XM_002748302XP_002748348 (SEQ ID NO: 28) (SEQ ID NO: 29) Rattus norvegicus NM_019259NP_062132 (SEQ ID NO: 30) (SEQ ID NO: 31) Mus musculus NM_007573NP_031599 (SEQ ID NO: 32) (SEQ ID NO: 33) Bos taurus NM_001034527NP_001029699 (SEQ ID NO: 34) (SEQ ID NO: 35) Felis catus XM_006939728XP_006939790 (SEQ ID NO: 36) (SEQ ID NO: 37) Canis lupus familiarisXM_546568 XP_546568 (SEQ ID NO: 38) (SEQ ID NO: 39) Equus caballusXM_001918118 XP_001918153 (SEQ ID NO: 40) (SEQ ID NO: 41) ¹Listed areexemplary GENBANK ® biosequence database Accession Nos.

As disclosed herein, the interaction of the exemplary TT1 Peptides ofSEQ ID NOs: 1-3 and gC1qR/p32 was identified. Based on these findings,disclosed herein are TT1 Family Peptide compositions useful in diseasesand disorders associated with gC1qR/p32. For example, the TT1 FamilyPeptide compositions disclosed herein are useful for reducing orpreventing tumor metastasis in cancer patients having a primary tumor.The TT1 Family Peptide compositions can be administered, for example, toa subject having pre-metastatic breast or bone cancer or to a subjecthaving early or late stage metastatic breast or bone cancer. TT1 FamilyPeptide polypeptides can also be useful, for example, for imaging tumorlymphatic vasculature, such as breast cancer or osteosarcoma lymphaticvasculature. The disclosed compositions are also useful for reducing orpreventing inflammation in patients in need thereof.

Thus, disclosed herein are isolated peptides or peptidomimeticcontaining the pentapeptide amino acid motif RGXRS (SEQ ID NO: 4), or apeptidomimetic thereof. The presently disclosed subject matter furtherprovides an isolated peptide or peptidomimetic containing the amino acidsequence KRGARST (SEQ ID NO: 1), the amino acid sequence AKRGARSTA (SEQID NO: 2), or the amino acid sequence CKRGARSTC (SEQ ID NO: 3) or apeptidomimetic thereof. Disclosed are compositions, such as thosecomprising a member of the TT1 Family of Peptides, that selectivelyinteract with tumors and sites of inflammation, as well as otherdiseases and disorders associated with gC1qR/p32. A variety of TT1Peptide compositions can be used in the disclosed methods. Suchcompositions include, without limitation, peptides as disclosed herein.The disclosed compounds, compositions, molecules, and methods caninclude or use the disclosed TT1 Peptide compositions in various forms,including peptides and peptidomimetics as disclosed. For convenience ofexpression, in many places herein the use or inclusion of peptides willbe recited. It is understood that, in such cases, it is considered thatTT1 Peptide compositions in various forms can also be used or includedin the same or similar ways as is described in terms of peptides, andsuch use and inclusion is specifically contemplated and disclosedthereby.

There are multiple diseases and disorders associated with the gC1q/p32receptor expression at the cell surface. Examples include, but are notlimited to, cancer, atherosclerosis, and inflammation.

The composition comprising members of the TT1 Family of Peptides canfurther comprise a moiety. Examples of moieties include, but are notlimited to, therapeutic or diagnostic moieties. Therapeutic moieties caninclude anti-angiogenic agents or cytotoxic agents. The therapeuticmoiety can target a DNA-associated process. The therapeutic moiety canbe selected from the group consisting of an alkylating agent, ananti-tumor antibiotic and a sequence-selective agent. Other examples oftherapeutic moieties include cyclophosphamide, melphalan, mitomycin C,bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone, SN-38,Et-743, actinomycin D, bleomycin, geldanamycin, chlorambucil,methotrexate, and TLK286. The moiety can also be a nanoparticle.

Disclosed are methods of detecting the presence of gC1q/p32 receptor,the method comprising bringing into contact a cell and a TT1 Peptidecomposition, wherein the TT1 Peptide composition comprises a moietylinked to a composition comprising the amino acid sequence KRGARST (SEQID NO: 1), the amino acid sequence AKRGARSTA (SEQ ID NO: 2), or theamino acid sequence CKRGARSTC (SEQ ID NO: 3); and detecting interactionbetween gC1q/p32 receptor and the TT1 Peptide composition, therebydetecting the presence of gC1q/p32 receptor. The gC1q/p32 receptor canbe, for example, on or in a cell. The cell can be in any context, suchas in an organism, in situ, ex vivo, in culture, and/or in vitro.

The moiety can be a detectable moiety. Examples of such moietiesinclude, but are not limited to, a polypeptide, a nucleic acid molecule,a small molecule, a fluorophore, fluorescein, rhodamine, a radionuclide,indium-111, technetium-99, carbon-11, carbon-13, or a combinationthereof.

The TT1 Peptide composition being brought into contact with the celldescribed above can comprise a virus in one example. The TT1 Peptidecomposition can also comprise a phage.

By “selectively interacts with” is meant that a stated compound ormaterial can preferentially interact with a stated target compared withnon-targets. Thus, for example, in vivo, a TT1 Peptide canpreferentially interact with the gC1qR/p32 as compared to non-target.

Therefore, when gC1qR/p32 is associated with a cancerous cell, or a siteof inflammation, a TT1 Peptide will interact with the cancerous cell orsite of inflammation preferentially, as compared to a non-cancerouscell, or a site without inflammation. Selective or preferentialinteraction with, for example, tumors, generally is characterized by atleast a two-fold or greater localization at the cancerous site. A TT1Peptide can be characterized by 5-fold, 10-fold, 20-fold, or morepreferential localization to cancerous sites such as tumors, as comparedto several or many tissue types of non-tumoral tissue, or as compared tomost or all non-tumoral tissue. Thus, it is understood that, in somecases, a TT1 Peptide interacts with, in part, one or more normal organsin addition to those with gC1qR/p32 present. Selective interaction canalso be referred to as targeting or homing.

As discussed above, selectively interacting with, including preferentialand/or selective homing, does not mean that a TT1 Peptide does not bindto any normal and/or non-targeted areas. In some embodiments,interaction selectivity can be, for example, at least about 20-fold, atleast about 30-fold, at least about 50-fold, at least about 75-fold, atleast about 100-fold, at least about 150-fold, or at least about200-fold selective for a corresponding target.

Selective interaction can be, for example, in terms of relative amountsor in terms of relative Ki over other non-target components. In someembodiments, a TT1 Peptide can have at least about a 50-foldselectivity, at least about a 100-fold selectivity, at least about a200-fold selectivity, at least about a 300-fold selectivity, at leastabout a 400-fold selectivity, at least about a 500-fold selectivity, atleast about a 600-fold selectivity, at least about a 700-foldselectivity, at least about an 800-fold selectivity, at least about a1000-fold selectivity, or at least about a 1500-fold selectivity to acorresponding target. For example, a TT1 Peptide can have a Ki valueagainst a target of in some embodiments less than about 200 nM, in someembodiments less than about 150 nM, in some embodiments less than about100 nM, or in some embodiments less than about 75 nM. A TT1 Peptide canhave a Ki value against a target of in some embodiments more than about50 nM, in some embodiments more than about 25 nM, in some embodimentsmore than about 20 nM, in some embodiments more than about 15 nM, insome embodiments more than about 10 nM, in some embodiments more thanabout 5 nM, in some embodiments more than about 3 nM, and in someembodiments more than about 1 nM. The targeting moiety can bind itstarget with a dissociation constant (k_(D)) in some embodiments lessthan about 10⁻⁸ M, in some embodiments less than about 10⁻⁹ M, in someembodiments less than about 10⁻¹⁰ M, in some embodiments less than about10⁻¹¹ M, in some embodiments less than about 10⁻¹² M, in someembodiments less than about 10⁻¹³ M, and in some embodiments less thanabout 10⁻¹⁴ M.

II.B. gC1q/p32 Receptor

It has been found that knocking down gC1qR/p32 expression in tumor cellsshifted their metabolism toward glycolysis and that, surprisingly, theglycolytic phenotype was associated with impaired tumor cell survivaland growth, especially under adverse growth conditions (see EXAMPLE 2 ofU.S. Pat. No. 8,178,104). At the same time, tumorigenicity of thegC1qR/p32 knockdown cells was reduced. Therefore, disclosed herein aremethods of targeting the gC1q/p32 receptor in order to treat gC1q/p32receptor-related disorders and diseases, as described herein. An exampleof such a disease is cancer.

Also disclosed herein is a method of treating a disease in a subjectassociated with gC1q/p32 receptor, the method comprising administeringto the subject a composition that modulates gC1q/p32 receptor expressionor activity, thereby treating a disease in a subject associated with thegC1q/p32 receptor. The subject can have cancer. Expression or activityof the gC1q/p32 receptor can be inhibited. This can occur by the use ofinterfering nucleic acid, such as shRNA or siRNA. In some embodiments,activity of the gC1q/p32 receptor can be inhibited by a TT1 Peptide, anantibody, and/or a small molecule mimic of a TT1 Peptide. The methods oftreating cancer disclosed herein can be used in conjunction with othertreatment therapies as well, as described below in the section relatingto moieties.

Disclosed herein are subjects having a disease associated with thegC1q/p32 receptor. By this is meant that in some embodiments the subjecthas either an increased level of gC1q/p32 receptor, a decreased level ofgC1q/p32 receptor, or that the gC1q/p32 receptor can be targeted totreat or ameliorate the symptoms of a disease or disorder. By an“increased level of gC1q/p32 receptor” is meant that the number ofgC1q/p32 receptors in the subject as a whole is increased over normal,basal, or standard levels accepted by those of skill in the art. It canalso mean that the number of gC1q/p32 receptors present in a given cellis increased over a basal, normal, or standard amount. By a “decreasedlevel of gC1q/p32 receptor” is meant that the number of gC1q/p32receptors in the subject as a whole is deceased over normal, basal, orstandard levels accepted by those of skill in the art. It can also meanthat the number of gC1q/p32 receptors present in a given cell aredecreased over a basal, normal, or standard amount. One of skill in theart would be able to determine gC1q/p32 levels in a subject as a whole,as well as in individual cells, using the methods disclosed herein andthose known to those of skill in the art. One method of doing soinvolves using a composition comprising a TT1 Peptide, as disclosedherein. Diseases associated with the gC1q/p32 receptor include cancer,for example. In some embodiments, the subject has an increased level ofgC1q/p32 receptor at the cell surface over normal (i.e., the same celltype in a subject that does not have the relevant disease or disorder),basal, or standard levels accepted by those of skill in the art,permitting the gC1q/p32 receptor to be targeted to treat or amelioratethe symptoms of the relevant disease or disorder.

II.C. Peptides and Peptidomimetics

Disclosed are compositions related to isolated TT1 Family Peptidescomprising in some embodiments the amino acid sequence KRGARST (SEQ IDNO: 1), the amino acid sequence AKRGARSTA (SEQ ID NO: 2), or the aminoacid sequence CKRGARSTC (SEQ ID NO: 3). The isolated peptides cancomprise, for example, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, anamino acid sequence at least about 90% identical to SEQ ID NO: 1, SEQ IDNO: 2, or SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 1, SEQID NO: 2, or SEQ ID NO: 3 having one or more conservative amino acidsubstitutions. The peptide can be at least about 90%, 80%, 70%, or 60%identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3 can have one, two, three, four, five, six, seven, eight, ornine conservative amino acid substitutions, for example. The peptide cancomprise a chimera of the amino acid sequence SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3. Such a chimera can be additive, where sequence ofone sequence is added to another sequence, substitutional, wheresequence of one sequence is substituted for sequence of anothersequence, or a combination. As used herein in reference to a specifiedamino acid sequence, a “conservative variant” is a sequence in which afirst amino acid is replaced by another amino acid or amino acid analoghaving at least one biochemical property similar to that of the firstamino acid; similar properties include, for example, similar size,charge, hydrophobicity or hydrogen-bonding capacity.

The amino acid sequence can be linear, circular, or cyclic. The aminoacid segment can be circularized or cyclized via any suitable linkage,for example, a disulfide bond.

The disclosed peptides can be in isolated form. As used herein inreference to the disclosed peptides, the term “isolated” means a peptidethat is in a form that is relatively free from material such ascontaminating polypeptides, lipids, nucleic acids, and other cellularmaterial that normally is associated with the peptide in a cell or thatis associated with the peptide in a library or in a crude preparation.

The disclosed peptides can have any suitable length. The disclosedpeptides can have, for example, a relatively short length of less thansix, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 45, or 50residues. The disclosed peptides also can be useful in the context of asignificantly longer sequence. Thus, the peptides can have, for example,a length of up to 75, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000residues. In some embodiments, a peptide can have a length of at least8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 residues. In someembodiments, a peptide can have a length of 5 to 200 residues, 5 to 100residues, 5 to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60residues, 5 to 50 residues, 5 to 40 residues, 5 to 30 residues, 5 to 20residues, 5 to 15 residues, 5 to 10 residues, 10 to 200 residues, 10 to100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues,10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30residues, 10 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues,20 to 50 residues, 20 to 40 residues or 20 to 30 residues. As usedherein, the term “residue” refers to an amino acid or amino acid analog.

As this specification discusses various proteins, protein sequences,peptides, peptides sequences, and amino acid sequences, it is understoodthat the nucleic acids that can encode those sequences are alsodisclosed. This would include all degenerate sequences related to aspecific protein sequence, i.e., all nucleic acids having a sequencethat encodes one particular protein sequence as well as all nucleicacids, including degenerate nucleic acids, encoding the disclosedvariants and derivatives of the protein sequences. Thus, while eachparticular nucleic acid sequence may not be written out herein, it isunderstood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. The disclosedpeptides and proteins can be coupled to each other via peptide bonds toform fusion peptides and proteins.

The disclosed peptides and amino acid segments can be modified. As usedherein, a “methylated derivative” of a protein, peptide, amino acidsegment, amino acid sequence, etc. refers to a form of the protein,peptide, amino acid segment, amino acid sequence, etc. that ismethylated. Unless the context indicates otherwise, reference to amethylated derivative of a protein, peptide, amino acid segment, aminoacid sequence, etc. does not include any modification to the baseprotein, peptide, amino acid segment, amino acid sequence, etc. otherthan methylation. Methylated derivatives can also have othermodifications, but such modifications generally will be noted. Forexample, conservative variants of an amino acid sequence would includeconservative amino acid substitutions of the based amino acid sequence.Thus, reference to, for example, a “methylated derivative” of a specificamino acid sequence “and conservative variants thereof” would includemethylated forms of the specific amino acid sequence and methylatedforms of the conservative variants of the specific amino acid sequence,but not any other modifications of derivations. As another example,reference to a methylated derivative of an amino acid segment thatincludes amino acid substitutions would include methylated forms of theamino acid sequence of the amino acid segment and methylated forms ofthe amino acid sequence of the amino acid segment include amino acidsubstitutions.

Protein variants and derivatives are well understood by those of skillin the art and in can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional, or deletionalvariants. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Insertions ordinarily will be smaller insertions than those ofamino or carboxyl terminal fusions, for example, on the order of one tofour residues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure.

As used herein in reference to a specified amino acid sequence, a“conservative variant” is a sequence in which a first amino acid isreplaced by another amino acid or amino acid analog having at least onebiochemical property similar to that of the first amino acid; similarproperties include, for example, similar size, charge, hydrophobicity orhydrogen-bonding capacity. Conservative variants are also referred toherein as “conservative amino acid substitutions,” “conservative aminoacid variants,” “conservative substitutions,” and similar phrase. A“conservative derivative” of a reference sequence refers to an aminoacid sequence that differs from the reference sequences only inconservative substitutions.

As an example, a conservative variant can be a sequence in which a firstuncharged polar amino acid is conservatively substituted with a second(non-identical) uncharged polar amino acid such as cysteine, serine,threonine, tyrosine, glycine, glutamine, or asparagine or an analogthereof. A conservative variant also can be a sequence in which a firstbasic amino acid is conservatively substituted with a second basic aminoacid such as arginine, lysine, histidine, 5-hydroxylysine,N-methyllysine, or an analog thereof. Similarly, a conservative variantcan be a sequence in which a first hydrophobic amino acid isconservatively substituted with a second hydrophobic amino acid such asalanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, or tryptophan or an analog thereof. In the same way, aconservative variant can be a sequence in which a first acidic aminoacid is conservatively substituted with a second acidic amino acid suchas aspartic acid or glutamic acid or an analog thereof; a sequence inwhich an aromatic amino acid such as phenylalanine is conservativelysubstituted with a second aromatic amino acid or amino acid analog, forexample, tyrosine; or a sequence in which a first relatively small aminoacid such as alanine is substituted with a second relatively small aminoacid or amino acid analog such as glycine or valine or an analogthereof. For example, the replacement of one amino acid residue withanother that is biologically and/or chemically similar is known to thoseskilled in the art as a conservative substitution. For example, aconservative substitution would be replacing one hydrophobic residue foranother, or one polar residue for another. The substitutions includecombinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu;Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservativelysubstituted variations of each explicitly disclosed sequence areincluded within the mosaic polypeptides provided herein. It isunderstood that conservative variants of the disclosed amino acidsequences can encompass sequences containing, for example, one, two,three, four or more amino acid substitutions relative to the referencesequence, and that such variants can include naturally and non-naturallyoccurring amino acid analogs.

Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place. Non-limitingexamples of such substitutions, referred to as conservativesubstitutions, can generally be made in accordance with the followingTable 2.

TABLE 2 Amino Acid Substitutions Original Amino Conservative AcidSubstitutions Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn;Lys Glu Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; GlnMet Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe ValIle; Leu

In some embodiments, a conservative substitution can be based on therelative similarity of the amino acid side-chain substituents. SeeHenikoff & Henikoff, 2000. Exemplary factors that can be considered inmaking conservative substitutions include side-chain hydrophobicity,hydrophilicity, charge, and size. For example, arginine, lysine, andhistidine are all positively charged residues; alanine, glycine, andserine are all of similar size; and phenylalanine, tryptophan, andtyrosine all have a generally similar shape. By this analysis, arginine,lysine, and histidine; alanine, glycine, and serine; and phenylalanine,tryptophan, and tyrosine; are defined herein to be conservativesubstitutions.

In making conservative substitutions, the hydropathic index of aminoacids can be considered. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine(+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan(−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate(−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine(−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982). It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are in some embodiments within ±2 of the originalvalue can be made, those that are in some embodiments within ±1 of theoriginal value can be made, and those that are in some embodimentswithin ±0.5 of the original value can be made.

It is also understood in the art that a conservative substitutions canbe made effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with its immunogenicity and antigenicity, e.g., with abiological property of the protein. It is understood that an amino acidcan be substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent protein.

Thus, and as detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

In making conservative substitutions based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arein some embodiments within +2 of the original value can be made, thosethat are in some embodiments within +1 of the original value can bemade, and those in some embodiments within +0.5 of the original valuecan be made.

Substantial changes in function or immunological identity can be made byselecting substitutions that are less conservative, i.e., selectingresidues that differ more significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site or (c) thebulk of the side chain. The substitutions which in general are expectedto produce the greatest changes in the protein properties will be thosein which (a) a hydrophilic residue, e.g., a seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine, in this case,(e) by increasing the number of sites for sulfation and/orglycosylation. These can be referred to as less conservative variants.

Peptides can have a variety of modifications. Modifications can be usedto change or improve the properties of the peptides. For example, thedisclosed peptides can be N-methylated, O-methylated, S-methylated,C-methylated, or a combination at one or more amino acids.

The amino and/or carboxy termini of the disclosed peptides can bemodified. Amino terminus modifications include methylation (e.g., —NHCH₃or —N(CH₃)₂), acetylation (e.g., with acetic acid or a halogenatedderivative thereof such as α-chloroacetic acid, α-bromoacetic acid, orα-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blockingthe amino terminus with any blocking group containing a carboxylatefunctionality defined by RCOO— or sulfonyl functionality defined byR—SO2-, where R is selected from the group consisting of alkyl, aryl,heteroaryl, alkyl aryl, and the like, and similar groups. One can alsoincorporate a desamino acid at the N-terminus (so that there is noN-terminal amino group) to decrease susceptibility to proteases or torestrict the conformation of the peptide compound. In preferredembodiments, the N-terminus is acetylated with acetic acid or aceticanhydride.

Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. One can also cyclize the disclosedpeptides, or incorporate a desamino or descarboxy residue at the terminiof the peptide, so that there is no terminal amino or carboxyl group, todecrease susceptibility to proteases or to restrict the conformation ofthe peptide. C-terminal functional groups of the disclosed peptidesinclude amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,hydroxy, and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

One can replace the naturally occurring side chains of the geneticallyencoded amino acids (or the stereoisomeric D amino acids) with otherside chains, for instance with groups such as alkyl, lower (C₁₋₆) alkyl,cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amidedi(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower esterderivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic. Inparticular, proline analogues in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, canbe aromatic or non-aromatic. Heterocyclic groups preferably contain oneor more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of suchgroups include the furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino),oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g.,1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. Theseheterocyclic groups can be substituted or unsubstituted. Where a groupis substituted, the substituent can be alkyl, alkoxy, halogen, oxygen,or substituted or unsubstituted phenyl.

One can also readily modify peptides by phosphorylation, and othermethods (e.g., as described in Hruby et al., 1990).

The disclosed peptides also serve as structural models for non-peptidiccompounds with similar biological activity. Those of skill in the artrecognize that a variety of techniques are available for constructingcompounds with the same or similar desired biological activity as thelead peptide compound, but with more favorable activity than the leadwith respect to solubility, stability, and susceptibility to hydrolysisand proteolysis (see Morgan & Gainor, 1989). These techniques include,but are not limited to, replacing the peptide backbone with a backbonecomposed of phosphonates, amidates, carbamates, sulfonamides, secondaryamines, and N-methylamino acids.

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO—. These andothers can be found in Spatola, 1983a; Spatola, 1983b (general review);Morley, 1980; Hudson et al., 1979 (—CH₂NH—, CH₂CH₂—); Spatola et al.,1986 (—CHH₂—S); Hann, 1982 (—CH—CH—, cis and trans); Almquist et al.,1980 (—COCH₂—); Jennings-White et al., 1982 (—COCH₂—); European PatentApplication Publication No. EP 0045665 (—CH(OH)CH₂—); Holladay et al.,1983 (—C(OH)CH₂—); and Hruby, 1982 (—CH₂—S—); each of which isincorporated herein by reference. An exemplary non-peptide linkage is—CH₂NH—. It is understood that peptide analogs can have more than oneatom between the bond atoms, such as β-alanine, γ-aminobutyric acid, andthe like.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also can be desirable.Deletions or substitutions of potential proteolysis sites, e.g., Arg,can be accomplished, for example, by deleting one of the basic residuesor substituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations can be the result of theaction of recombinant host cells on the expressed polypeptide.Glutaminyl and asparaginyl residues are frequently post-translationallydeamidated to the corresponding glutamyl and asparyl residues.Alternatively, these residues are deamidated under mildly acidicconditions. Other post-translational modifications include hydroxylationof proline and lysine, phosphorylation of hydroxyl groups of seryl orthreonyl residues, methylation of the o-amino groups of lysine,arginine, and histidine side chains (Creighton, 1983), acetylation ofthe N-terminal amine and, in some instances, amidation of the C-terminalcarboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed amino acids sequences, amino acid segments, peptides,proteins, etc. herein is through defining the variants and derivativesin terms of homology/identity to specific known sequences. For example,specifically disclosed are variants of these and other amino acidssequences, amino acid segments, peptides, proteins, etc. hereindisclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95%homology to the stated sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins. For example,the homology can be calculated after aligning the two sequences so thatthe homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison can beconducted by the local homology algorithm of Smith & Waterman, 1981, bythe homology alignment algorithm of Needleman & Wunsch, 1970, by thesearch for similarity method of Pearson & Lipman, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis., United States of America), or by inspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, 1989; Jaeger et al., 1989a;Jaeger et al., 1989b1 each of which is herein incorporated by referencefor at least material related to nucleic acid alignment.

It is understood that the description of conservative variants andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative variants.

As this specification discusses various amino acids sequences, aminoacid segment sequences, peptide sequences, protein sequences, etc., itis understood that nucleic acids that can encode those sequences arealso disclosed. This would include all degenerate sequences related to aspecific amino acid sequence, i.e. all nucleic acids having a sequencethat encodes one particular amino acid sequence as well as all nucleicacids, including degenerate nucleic acids, encoding the disclosedvariants and derivatives of the amino acid sequences. Thus, while eachparticular nucleic acid sequence may not be written out herein, it isunderstood that each and every sequence is in fact disclosed anddescribed herein through the disclosed amino acid sequences.

Also disclosed are bifunctional peptides, which contain a TT1 Peptidefused to a second peptide having a separate function. Such bifunctionalpeptides have at least two functions conferred by different portions ofthe full-length molecule and can, for example, display anti-angiogenicactivity or pro-apoptotic activity in addition to the ability toselectively interact with gC1qR/p32.

Also disclosed are isolated multivalent peptides that include at leasttwo subsequences each independently containing a peptide (for example,the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3,or a conservative variant or peptidomimetic thereof). The multivalentpeptide can have, for example, at least three, at least five, or atleast ten of such subsequences each independently containing a peptide.In particular embodiments, the multivalent peptide can have two, three,four, five, six, seven, eight, nine, ten, fifteen, or twenty identicalor non-identical subsequences. In a further embodiment, the multivalentpeptide can contain identical subsequences, such as repeats of SEQ IDNO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3. In a further embodiment, themultivalent peptide contains contiguous identical or non-identicalsubsequences, which are not separated by any intervening amino acids. Inyet further embodiments, the multivalent peptide can be cyclic orotherwise conformationally constrained. In one example, the peptide canbe circularized or cyclized via a disulfide bond.

As used herein, the term “peptide” is used broadly to mean peptides,proteins, fragments of proteins and the like. The term “peptidomimetic”,as used herein, means a peptide-like molecule that has the activity ofthe peptide upon which it is structurally based. Such peptidomimeticsinclude chemically modified peptides, peptide-like molecules containingnon-naturally occurring amino acids, and peptoids and have an activitysuch as selective interaction with a target of the peptide upon whichthe peptidomimetic is derived (see e.g., Goodman & Ro, 1995.

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; α,α-dialkylglycine or α-aminocycloalkanecarboxylic acid; an N^(α)-C^(α) cyclized amino acid; an N^(α)-methylatedamino acid; β- or γ-amino cycloalkane carboxylic acid; an α,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2, 3-methano amino acid; an N—C^(€) or C^(α)-C^(Δ)cyclized amino acid; a substituted proline or another amino acidmimetic. A peptidomimetic which mimics peptide secondary structure cancontain, for example, a non-peptidic β-turn mimic; γ-tum mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylene-sulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. As an example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (see Allen et al., 1979). This structuraldepository is continually updated as new crystal structures aredetermined and can be screened for compounds having suitable shapes, forexample, the same shape as a disclosed peptide, as well as potentialgeometrical and chemical complementarity to a target molecule. Where nocrystal structure of a peptide or a target molecule that binds thepeptide is available, a structure can be generated using, for example,the program CONCORD (Rusinko et al., 1989). Another database, theAvailable Chemicals Directory (Molecular Design Limited, InformationSystems; San Leandro, Calif., United States of America), contains about100,000 compounds that are commercially available and also can besearched to identify potential peptidomimetics of a peptide, forexample, with activity in selectively interacting with cancerous cells.

Also disclosed are chimeric proteins containing a disclosed peptidefused to a heterologous protein. In some embodiments, the heterologousprotein can have a therapeutic activity such as cytokine activity,cytotoxic activity or pro-apoptotic activity. In a further embodiment,the heterologous protein can be an antibody or antigen-binding fragmentthereof. In other embodiments, the chimeric protein includes a peptidecontaining the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3, or a conservative variant or peptidomimetic thereof, fused toa heterologous protein. The term “heterologous,” as used herein inreference to a protein fused to the disclosed peptides, means a proteinderived from a source other than the gene encoding the peptide or fromwhich the peptidomimetic is derived. The disclosed chimeric proteins canhave a variety of lengths including, but not limited to, a length ofless than 100 residues, less than 200 residues, less than 300 residues,less than 400 residues, less than 500 residues, less than 800 residues,less than 1000 residues, or greater than 1000 residues.

As used herein, “chimera” and “chimeric” refer to any combination ofsequences derived from two or more sources. This includes, for example,from single moiety of subunit (e.g., nucleotide, amino acid) up toentire source sequences added, inserted, and/or substituted into othersequences. Chimeras can be, for example, additive, where one or moreportions of one sequence are added to one or more portions of one ormore other sequences; substitutional, where one or more portions of onesequence are substituted for one or more portions of one or more othersequences; or a combination. “Conservative substitutional chimeras” canbe used to refer to substitutional chimeras where the source sequencesfor the chimera have some structural and/or functional relationship andwhere portions of sequences having similar or analogous structure and/orfunction are substituted for each other. Typical chimeric and humanizedantibodies are examples of conservative substitutional chimeras.

If desired, an isolated peptide such as a TT1 Peptide can be cyclic orotherwise conformationally constrained. As used herein, a“conformationally constrained” molecule, such as a peptide, is one inwhich the three-dimensional structure is maintained substantially in onespatial arrangement over time. Conformationally constrained moleculescan have improved properties such as increased affinity, metabolicstability, membrane permeability, or solubility. Methods ofconformational constraint are well known in the art and includecyclization as discussed further elsewhere herein.

As used herein in reference to a peptide, the term “cyclic” means astructure including an intramolecular bond between two non-adjacentamino acids or amino acid analogues. The cyclization can be effectedthrough a covalent or non-covalent bond. Intramolecular bonds include,but are not limited to, backbone to backbone, side-chain to backbone andside-chain to side-chain bonds. A preferred method of cyclization isthrough formation of a disulfide bond between the side-chains ofnon-adjacent amino acids or amino acid analogs. Residues capable offorming a disulfide bond include, for example, cysteine (Cys),penicillamine (Pen), pentamethylene cysteine (Pmc),β,β-pentamethylene-β-mercaptopropionic acid (Pmp), and functionalequivalents thereof.

A peptide also can cyclize, for example, via a lactam bond, which canutilize a side-chain group of one amino acid or analog thereof to form acovalent attachment to the N-terminal amine of the amino-terminalresidue. Residues capable of forming a lactam bond include aspartic acid(Asp), glutamic acid (Glu), lysine (Lys), ornithine (om),α,β-diamino-propionic acid, γ-amino-adipic acid (Adp) andM-(aminomethyl)benzoic acid (Mamb). Cyclization additionally can beeffected, for example, through the formation of a lysinonorleucine bondbetween lysine (Lys) and leucine (Leu) residues or a dityrosine bondbetween two tyrosine (Tyr) residues. The skilled person understands thatthese and other bonds can be included in a cyclic peptide.

II.D. TT1 Peptide Compositions Comprising Moieties

The TT1 Peptide compositions disclosed herein are in some embodimentsTT1 Peptide compositions comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3, which optionally can further comprise a moiety. The moiety canbe any molecule. For example, disclosed are moieties containing atherapeutic agent linked to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.In some embodiments, the moiety is a molecule that is usefully targetedto the gC1q/p32 receptor. For example, moieties that affect the target,such as moieties with therapeutic effect, or that facilitate detection,visualization, or imaging of the target, such as fluorescent molecule orradionuclides. The disclosed peptides, such as SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3, that selectively interact with gC1qR/p32 can beusefully combined with, for example, moieties that can, for example,affect tumors and cancer, reduce or eliminate inflammation or infection,and/or promote wound healing. A variety of therapeutic agents are usefulin the TT1 Peptide compositions disclosed herein, including, withoutlimitation, cancer chemotherapeutic agents, cytotoxic agents,anti-angiogenic agents, polypeptides, nucleic acid molecules, and smallmolecules.

A TT1 Peptide composition can comprise, for example, two or more, threeor more, five or more, ten or more, twenty or more, thirty or more,forty or more, fifty or more, 100 or more, 200 or more, 300 or more, 400or more, 500 or more, or 1000 or more copies of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3. The TT1 Peptide composition can comprise peptidesthat all have an identical amino acid sequence. In some embodiments, theTT1 Peptide composition can comprise two or more non-identical aminoacid sequences. For example, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3and another targeting peptide can be used separately or together in aTT1 Peptide composition of the presently disclosed subject matter.Moieties useful in a TT1 Peptide composition incorporating multiplepeptides include, without limitation, phage, retroviruses, adenoviruses,adeno-associated viruses and other viruses, cells, liposomes, polymericmatrices, non-polymeric matrices, particles such as gold particles,microdevices, nanodevices, and nanoscale semiconductor materials.

A TT1 Peptide composition can contain, for example, a liposome or otherpolymeric matrix linked to at least two peptides. If desired, theliposome or other polymeric matrix can be linked to at least ten, atleast 100 or at least 1000 peptides such as SEQ ID NO: 1, SEQ ID NO: 2,or SEQ ID NO: 3. Liposomes can be useful in such conjugates; liposomesconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer (see e.g., Gregoriadis, 1984). The liposome or otherpolymeric matrix can optionally include another component such as,without limitation, a therapeutic agent, cancer chemotherapeutic agent,cytotoxic agent, anti-angiogenic agent, and polypeptide or nucleic acidmolecule.

Components of the disclosed TT1 Peptide compositions can be combined,linked, and/or coupled in any suitable manner. By way of example and notlimitation, moieties and peptides can be associated covalently ornon-covalently, directly or indirectly, with or without a linker moiety.

In some embodiments, one or more of the homing molecules can comprise,consist essentially of, and/or consist of the amino acid sequenceKRGARST (SEQ ID NO: 1), the amino acid sequence AKRGARSTA (SEQ ID NO:2), or a conservative derivative thereof, the amino acid sequenceCKRGARSTC (SEQ ID NO: 3) or a conservative derivative thereof, or acombination. In some embodiments, one or more of the homing molecule cancomprise, consist essentially of, and/or consist of the amino acidsequence KRGARST (SEQ ID NO: 1) or a conservative variant thereof. Insome embodiments, one or more of the homing molecules can comprise,consist essentially of, and/or consist of the amino acid sequenceKRGARST (SEQ ID NO: 1). In some embodiments, one or more of the membraneperturbing molecules can comprise, consist essentially of, and/orconsist of the amino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42) or aconservative variant thereof, (KLAKLAK)₂ (SEQ ID NO: 42) or aconservative variant thereof, (KLAKKLA)₂ (SEQ ID NO: 43) or aconservative variant thereof, (KAAKKAA)₂ (SEQ ID NO: 44) or aconservative variant thereof, and/or (KLGKKLG)₃ (SEQ ID NO: 45) or aconservative variant thereof, or a combination. In some embodiments, oneor more of the membrane perturbing molecules can comprise, consistessentially of, and/or consist of the amino acid sequence _(D)(KLAKLAK)₂(SEQ ID NO: 42), (KLAKLAK)₂ (SEQ ID NO: 42), (KLAKKLA)₂ (SEQ ID NO: 43),(KAAKKAA)₂ (SEQ ID NO: 44), (KLGKKLG)₃ (SEQ ID NO: 45), or a combinationthereof. In some embodiments, one or more of the membrane perturbingmolecules can comprise, consist essentially of, and/or consist of theamino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42) or a conservativevariant thereof. In some embodiments, one or more of the membraneperturbing molecules can comprise, consist essentially of, and/orconsist of the amino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42).

In some embodiments, the composition can comprise a plurality of surfacemolecules, a plurality of homing molecules, and a plurality of cargomolecules. In some embodiments, the composition can comprise one or moresurface molecules, a plurality of homing molecules and a plurality ofcargo molecules. In some embodiments, the composition can comprise aplurality of surface molecules, one or more homing molecules, and aplurality of cargo molecules. In some embodiments, the composition cancomprise a plurality of surface molecules, a plurality of homingmolecules, and one or more cargo molecules. In some embodiments, thecomposition can comprise one or more surface molecules, one or morehoming molecules, and a plurality of cargo molecules. In someembodiments, the composition can comprise one or more surface molecules,a plurality of homing molecules and one or more cargo molecules. In someembodiments, the composition comprises a plurality of surface molecules,one or more homing molecules, and one or more cargo molecules.

In some embodiments, the composition can comprise a surface molecule, aplurality of homing molecules and a plurality of cargo molecules,wherein one or more of the homing molecules and one or more of the cargomolecules are associated with the surface molecule. In some embodiments,the composition can comprise a surface molecule, a plurality of homingmolecules, and a plurality of cargo molecules, wherein a plurality ofthe plurality of homing molecules and a plurality of the plurality ofcargo molecules are associated with the surface molecule. In someembodiments, the composition can comprise a surface molecule, aplurality of homing molecules, and a plurality of cargo molecules,wherein the homing molecules and the cargo molecules are associated withthe surface molecule.

In some embodiments, the composition can comprise a surface molecule,wherein the surface molecule is multivalent for homing molecules andcargo molecules. In some embodiments, the composition can comprise asurface molecule, wherein the surface molecule is multivalent for homingmolecules and comprises one or more cargo molecules. In someembodiments, the composition can comprise a surface molecule, whereinthe surface molecule is multivalent for cargo molecules and comprisesone or more homing molecules. In some embodiments, the composition cancomprise a surface molecule, wherein the surface molecule is multivalentfor conjugates, wherein one or more of the conjugates comprise one ormore homing molecules and one or more cargo molecules. In someembodiments, the composition can comprise a surface molecule, whereinthe surface molecule is multivalent for conjugates, wherein one or moreof the conjugates comprise a plurality of homing molecules and aplurality of cargo molecules. In some embodiments, the composition cancomprise a surface molecule, wherein the surface molecule is multivalentfor conjugates, wherein one or more of the conjugates comprise a homingmolecule and a cargo molecule. In some embodiments, the composition cancomprise a surface molecule, wherein the surface molecule is multivalentfor conjugates, wherein each of the conjugates comprises a plurality ofhoming molecules and a plurality of cargo molecules. In someembodiments, the composition can comprise a surface molecule, whereinthe surface molecule is multivalent for conjugates, wherein each of theconjugates comprises a homing molecule and a cargo molecule. As usedherein, a component that is stated to be “multivalent for” one or moreother components refers to a component that has a plurality of the othercomponents associated with, conjugated to and/or covalent coupled to thefirst component.

In some embodiments, the composition can comprise a surface molecule,wherein the surface molecule comprises one or more conjugates, whereinone or more of the conjugates comprise one or more homing molecules andone or more cargo molecules. In some embodiments, the composition cancomprise a surface molecule, wherein the surface molecule comprises oneor more conjugates, wherein one or more of the conjugates comprise aplurality of homing molecules and a plurality of cargo molecules. Insome embodiments, the composition can comprise a surface molecule,wherein the surface molecule comprises one or more conjugates, whereinone or more of the conjugates comprise a homing molecule and a cargomolecule. In some embodiments, the composition can comprise a surfacemolecule, wherein the surface molecule comprises one or more conjugates,wherein each of the conjugates comprises a plurality of homing moleculesand a plurality of cargo molecules.

In some embodiments, the composition can comprise a surface molecule,wherein the surface molecule comprises one or more conjugates, whereineach of the conjugates comprises a homing molecule and a cargo molecule.

In some embodiments, one or more of the membrane perturbing moleculescan be conjugated to one or more of the homing molecules. In someembodiments, one or more of the conjugated membrane perturbing moleculesand homing molecules can be covalently coupled. In some embodiments, oneor more of the covalently coupled membrane perturbing molecules andhoming molecules can comprise fusion peptides. In some embodiments, thehoming molecules can be conjugated with the surface molecule. In someembodiments, one or more of the conjugated homing molecules can bedirectly conjugated to the surface molecule. In some embodiments, one ormore of the conjugated homing molecules can be indirectly conjugated tothe surface molecule. In some embodiments, one or more of the homingmolecules can be covalently coupled to the surface molecule. In someembodiments, one or more of the covalently coupled homing molecules canbe directly covalently coupled to the surface molecule. In someembodiments, one or more of the covalently coupled homing molecules canbe indirectly covalently coupled to the surface molecule. In someembodiments, the membrane perturbing molecules can be conjugated withthe surface molecule. In some embodiments, one or more of the conjugatedmembrane perturbing molecules are directly conjugated to the surfacemolecule. In some embodiments, one or more of the conjugated membraneperturbing molecules can be indirectly conjugated to the surfacemolecule. In some embodiments, one or more of the membrane perturbingmolecules can be covalently coupled to the surface molecule. In someembodiments, one or more of the covalently coupled membrane perturbingmolecules can be directly covalently coupled to the surface molecule. Insome embodiments, one or more of the covalently coupled membraneperturbing molecules can be indirectly covalently coupled to the surfacemolecule.

In some embodiments, the composition can further comprise one or moreinternalization elements. In some embodiments, one or more of the homingmolecules can comprise one or more of the internalization elements. Insome embodiments, one or more of the membrane perturbing molecules cancomprise one or more of the internalization elements. In someembodiments, the surface molecule can comprise one or more of theinternalization elements not comprised in either the homing molecules orthe membrane perturbing molecules. In some embodiments, the compositioncan further comprise one or more tissue penetration elements. In someembodiments, one or more of the tissue penetration elements can becomprised in an internalization element. In some embodiments, the tissuepenetration element can be a CendR element.

In some embodiments, the surface molecule can comprise a nanoparticle.In some embodiments, the surface molecule can comprise a nanoworm. Insome embodiments, the surface molecule can comprise an iron oxidenanoworm. In some embodiments, the surface molecule can comprise an ironoxide nanoparticle. In some embodiments, the surface molecule cancomprise an albumin nanoparticle. In some embodiments, the surfacemolecule can comprise a liposome. In some embodiments, the surfacemolecule can comprise a micelle. In some embodiments, the surfacemolecule comprises a phospholipid. In some embodiments, the surfacemolecule comprises a polymer. In some embodiments, the surface moleculecan comprise a microparticle. In some embodiments, the surface moleculecan comprise a fluorocarbon microbubble.

In some embodiments, the composition can comprise at least 100 homingmolecules. In some embodiments, the composition can comprise at least1000 homing molecules. In some embodiments, the composition can compriseat least 10,000 homing molecules. In some embodiments, the compositioncan comprise at least 100 membrane perturbing molecules. In someembodiments, the composition can comprise at least 1000 membraneperturbing molecules. In some embodiments, the composition can compriseat least 10,000 membrane perturbing molecules.

In some embodiments, one or more of the homing molecules can be modifiedhoming molecules. In some embodiments, one or more of the homingmolecules can comprise a methylated homing molecule. In someembodiments, one or more of the methylated homing molecules can comprisea methylated amino acid segment. In some embodiments, one or more of themembrane perturbing molecules can be modified membrane perturbingmolecules. In some embodiments, one or more of the membrane perturbingmolecules comprise a methylated membrane perturbing molecule. In someembodiments, one or more of the methylated membrane perturbing moleculescomprise a methylated amino acid segment.

In some embodiments, the amino acid sequence is N- or C-methylated in atleast one position.

The disclosed components can be associated with each other (or, in someembodiments, not associated with each other) in combinations asdisclosed herein. For example, homing molecules can be covalentlycoupled or non-covalently associated with surface molecules, homingmolecules can be covalently coupled or non-covalently associated withmembrane perturbing molecules, membrane perturbing molecules can becovalently coupled or non-covalently associated with surface molecules,etc. Associated components can also be referred to as being conjugated.Conjugation can be direct or indirect. Direct conjugation of componentsrefers to covalently coupled or non-covalently associated componentswhere there is no other molecule intervening between the conjugatedcomponents. Indirect conjugation refers to any chain of molecules andcovalent bonds or non-covalent associations linking the components wherethe components are not directly conjugated (that is, there is a leastone separate molecule other than the components intervening between thecomponents).

Covalently coupled refers to association of components via covalentbonds. A covalent association or coupling can be either direct orindirect. A direct covalent association or coupling of components refersto a covalent bond involving atoms that are each respectively a part ofthe components. Thus, in a direct covalent association or coupling,there is no other molecule intervening between the associated/coupledcomponents. An indirect covalent association or coupling refers to anychain of molecules and covalent bonds linking the components where thecomponents are not covalently coupled (that is, there is a least oneseparate molecule other than the components intervening between thecomponents via covalent bonds).

As used herein, reference to components (such as a homing molecule and asurface molecule) as being “not covalently coupled” means that thecomponents are not connected via covalent bonds (for example, that thehoming molecule and the surface molecule are not connected via covalentbonds). That is, there is no continuous chain of covalent bonds between,for example, the homing molecule and the surface molecule.

Non-covalent association refers to association of components vianon-covalent bonds and interactions. A non-covalent association can beeither direct or indirect. A direct non-covalent association refers to anon-covalent bond involving atoms that are each respectively connectedvia a chain of covalent bonds to the components. Thus, in a directnon-covalent association, there is no other molecule intervening betweenthe associated components. An indirect non-covalent association refersto any chain of molecules and bonds linking the components where thecomponents are not covalently coupled (that is, there is a least oneseparate molecule other than the components intervening between thecomponents via non-covalent bonds).

Reference to components (such as a homing molecule and a surfacemolecule) as not being “non-covalently associated” means that there isno direct or indirect non-covalent association between the components.That is, for example, no atom covalently coupled to a homing molecule isinvolved in a non-covalent bond with an atom covalently coupled to asurface molecule. Within this meaning, a homing molecule and a surfacemolecule can be together in a composition where they are indirectlyassociated via multiple intervening non-covalent bonds while not beingnon-covalently associated as that term is defined herein. For example, ahoming molecule and a surface molecule can be mixed together in acarrier where they are not directly non-covalently associated. A homingmolecule and a surface molecule that are referred to as not indirectlynon-covalently associated cannot be mixed together in a continuouscomposition. Reference to components (such as a homing molecule and asurface molecule) as not being “directly non-covalently associated”means that there is no direct non-covalent association between thecomponents (an indirect non-covalent association may be present).Reference to components (such as a homing molecule and a surfacemolecule) as not being “indirectly non-covalently associated” means thatthere is no direct or indirect non-covalent association between thecomponents.

It is understood that components can be non-covalently associated viamultiple chains and paths including both direct and indirectnon-covalent associations. For the purposes of these definitions, thepresence a single direct non-covalent association makes the associationa direct non-covalent association even if there are also indirectnon-covalent associations present. Similarly, the presence of a covalentconnection between components means the components are covalentlycoupled even if there are also non-covalent associations present. It isalso understood that covalently coupled components that happened to lackany non-covalent association with each other are not considered to fallunder the definition of components that are not non-covalentlyassociated.

Association of the components of the disclosed compositions can be aidedor accomplished via molecules, conjugates, and/or compositions. Wheresuch molecules, conjugates and/or compositions are other than surfacemolecules, homing molecules, or cargo molecules (such as membraneperturbing molecules, internalization elements, tissue penetrationelements, and moieties), they can be referred to herein as linkers. Suchlinkers can be any molecule, conjugate, composition, etc. that can beused to associate components of the disclosed compositions. Generally,linkers can be used to associate components other than surface moleculesto surface molecules. Useful linkers include materials that arebiocompatible, have low bioactivity, have low antigenicity, etc. Thatis, such useful linker materials can serve the linking/associationfunction without adding unwanted bioreactivity to the disclosedcompositions. Many such materials are known and used for similar linkingand association functions. Polymer materials are a particularly usefulform of linker material. For example, polyethylene glycols can be used.

Linkers are useful for achieving useful numbers and densities of thecomponents (such as homing molecules and membrane perturbing molecules)on surface molecules. For example, linkers of fibrous form are usefulfor increasing the number of components per surface molecule or per agiven area of the surface molecule. Similarly, linkers having abranching form are useful for increasing the number of components persurface molecule or per a given area of the surface molecule. Linkerscan also have a branching fibrous form.

Sufficiency of the number and composition of homing molecules in thecomposition can be determined by assessing homing to the target andeffectively delivery of the cargo molecules in a non-human animal. Thecomposition can comprise a sufficient number and composition of homingmolecules (modified or not) such that the composition homes to thetarget and effectively delivers the cargo molecules. In one example,sufficiency of the number and composition of modified and/or unmodifiedhoming molecules can be determined by assessing cargo delivery and/ortherapeutic effect on the target. Sufficiency of the number andcomposition of membrane perturbing molecules can be determined byassessing membrane perturbing effect of the composition in a non-humananimal. The composition can comprise a sufficient number and compositionof membrane perturbing molecules (modified or not) such that thecomposition has a membrane perturbing effect on the target. In oneexample, sufficiency of the number and composition of modified and/orunmodified membrane perturbing molecules can be determined by assessingmembrane disruption, apoptosis, and/or therapeutic effect on the target.

The composition can comprise a sufficient density and composition ofhoming molecules such that the composition homes to the target andeffectively delivers the cargo molecules. Sufficiency of the density andcomposition of homing molecules can be determined by assessing cargodelivery and/or therapeutic effect on the target in a non-human animal.The composition can comprise a sufficient density and composition ofmembrane perturbing molecules such that the composition has a membraneperturbing effect on the target. Sufficiency of the density andcomposition of membrane perturbing molecules can be determined byassessing membrane disruption, apoptosis, and/or therapeutic effect onthe target in a non-human animal.

The density of homing molecules and/or membrane perturbing molecules ona surface molecule can be described in any suitable manner. For example,the density can be expressed as the number of homing molecules and/ormembrane perturbing molecules per, for example, a given area, surfacearea, volume, unit, subunit, arm, etc. of the surface molecule. Thedensity can also be relative to, for example, the area, surface area,volume, unit, subunit, arm, etc. of the entire surface molecule or tothe area, surface area, volume, unit, subunit, arm, etc. of a portion ofthe surface molecule. For example, a sufficient density of homingmolecule and/or membrane perturbing molecule can be present in a portionof the surface molecule.

The presence of this dense portion can cause clotting and amplify theaccumulation of the composition. Thus, a composition having a sufficientdensity of homing molecules and/or membrane perturbing molecules canhave a threshold density (or above) for the entire surface molecule orfor just one or more portions of the surface molecule. Unless otherwisestated, densities refer to average density over the designated portionof the surface molecule. For example, a density of 1 homing molecule persquare nM of the surface molecule refers to an average density of thehoming molecules over the entire surface molecule. As another example, adensity of 1 homing molecule per square nM of a portion of the surfacemolecule refers to an average density of the homing molecules over justthat portion of the surface molecule.

The density can be measured or calculated in any suitable manner. Forexample, the number or amount of homing molecules and/or membraneperturbing molecules present on a surface molecule or group of surfacemolecules can be measured by, for example, detecting the level orintensity of signal produced by labeled homing molecules and/or membraneperturbing molecules and calculating the density based on the structuralcharacteristics of the surface molecule.

The density or threshold density of homing molecules and/or membraneperturbing molecules can be, for example, at least 0.001, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400,420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1000 homing molecules and/or membrane perturbing molecules per square nMof the entire or a portion of the surface molecule. The composition canalso comprise any density in between those densities listed above.

The density or threshold density of homing molecules and/or membraneperturbing molecules can be, for example, at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 900, 9500, 10,000 homing moleculesand/or membrane perturbing molecules per square μM of the entire or aportion of the surface molecule. The composition can also comprise anydensity in between those densities listed above.

The density or threshold density of homing molecules and/or membraneperturbing molecules can be, for example, at least 0.001, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400,420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1000 homing molecules and/or membrane perturbing molecules per cubic nMof the entire or a portion of the surface molecule. The composition canalso comprise any density in between those densities listed above.

The density or threshold density of homing molecules and/or membraneperturbing molecules can be, for example, at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 900, 9500, 10,000 homing moleculesand/or membrane perturbing molecules per cubic LM of the entire or aportion of the surface molecule. The composition can also comprise anydensity in between those densities listed above.

The number of homing molecules and/or membrane perturbing molecules on asurface molecule can be described in any suitable manner. For example,the number can be expressed as the number of homing molecules and/ormembrane perturbing molecules per, for example, a given area, surfacearea, volume, unit, subunit, arm, etc. of the surface molecule. Thenumber can also be relative to, for example, the area, surface area,volume, unit, subunit, arm, etc. of the entire surface molecule or tothe area, surface area, volume, unit, subunit, arm, etc. of a portion ofthe surface molecule. For example, a sufficient number of homingmolecule and/or membrane perturbing molecule can be present in a portionof the surface molecule. The presence of this dense portion can causeclotting and amplify the accumulation of the composition. Thus, acomposition having a sufficient number of homing molecules and/ormembrane perturbing molecules can have a threshold number (or above) forthe entire surface molecule or for just one or more portions of thesurface molecule.

The number can be measured or calculated in any suitable manner. Forexample, the number or amount of homing molecules and/or membraneperturbing molecules present on a surface molecule or group of surfacemolecules can be measured by, for example, detecting the level orintensity of signal produced by labeled homing molecules and/or membraneperturbing molecules and calculating the number based on the structuralcharacteristics of the surface molecule.

The number or threshold number of homing molecules and/or membraneperturbing molecules can be, for example, at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 900, 9500, 10,000 homing moleculesand/or membrane perturbing molecules on the surface molecule. Thecomposition can also comprise any number in between those numbers listedabove.

The number or threshold number of homing molecules and/or membraneperturbing molecules can be, for example, at least 0.001, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400,420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1000 homing molecules and/or membrane perturbing molecules per square nMof the entire or a portion of the surface molecule. The composition canalso comprise any number in between those numbers listed above.

The number or threshold number of homing molecules and/or membraneperturbing molecules can be, for example, at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 900, 9500, 10,000 homing moleculesand/or membrane perturbing molecules per square M of the entire or aportion of the surface molecule. The composition can also comprise anynumber in between those numbers listed above.

The number or threshold number of homing molecules and/or membraneperturbing molecules can be, for example, at least 0.001, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400,420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1000 homing molecules and/or membrane perturbing molecules per cubic nMof the entire or a portion of the surface molecule. The composition canalso comprise any number in between those numbers listed above.

The number or threshold number of homing molecules and/or membraneperturbing molecules can be, for example, at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 900, 9500, 10,000 homing moleculesand/or membrane perturbing molecules per cubic μM of the entire or aportion of the surface molecule. The composition can also comprise anynumber in between those numbers listed above.

In some embodiments, the compositions not only home to tumors, but alsoamplify their own homing. Homing molecules can be used that areclot-binding compounds that recognize clotted plasma proteins andselectively homes to tumors, where it binds to vessel walls and tumorstroma. Surface molecules coupled with the clot-binding compounds canaccumulate in tumor vessels or at wound sites, where they induceadditional local clotting, thereby producing new binding sites for moreparticles. The system mimics platelets, which also circulate freely butaccumulate at a diseased site and amplify their own accumulation at thatsite. The clotting-based amplification greatly enhances cargo deliveryand tumor imaging.

II.D.1. Homing Molecules

Homing molecules allow the disclosed compositions to be targeted and tohome to desired target sites. Homing molecules generally bindpreferentially to target molecules, cells, tissues, etc., thus resultingin an accumulation of the homing molecules (and other components towhich they are associated) at target sites.

The term “homing molecule” as used herein, means any molecule thatselectively homes in vivo to specified target sites, such as cells ortissues, in preference to normal or other non-target sites, cells, ortissues. Similarly, the term “homing peptide” or “homing peptidomimetic”means a peptide that selectively homes in vivo to specified targetsites, such as cells or tissues, in preference to normal or othernon-target sites, cells, or tissues. It is understood that a homingmolecule that selectively homes in vivo to, for example, tumors can hometo all tumors or can exhibit preferential homing to one or a subset oftumor types.

By “selectively homes” it is meant that in vivo, the homing moleculebinds preferentially to the target as compared to non-target. Forexample, the homing molecule can bind preferentially to certainmolecules, proteins, cells, tissues, etc. as compared to othermolecules, proteins, cells, tissues, etc. For example, the homingmolecule can bind preferentially to tumor vasculature or one or moretumors as compared to non-tumoral tissue.

Such a homing molecule can selectively home, for example, to tumors.Selective homing to, for example, certain molecules, proteins, cells,tissues, etc. generally is characterized by at least a two-fold greaterlocalization the molecules, proteins, cells, tissues, etc. (or othertarget), as compared to other certain molecules, proteins, cells,tissues, etc. A homing molecule can be characterized by, for example,5-fold, 10-fold, 20-fold or more preferential localization to the targetas compared to one or more non-targets. For example, a homing moleculecan be characterized by, for example, 5-fold, 10-fold, 20-fold or morepreferential localization to tumor vasculature as compared tovasculature of several or many tissue types of non-tumoral tissue, or ascompared to vasculature of most or all non-tumoral tissue. As anotherexample, a homing molecule can be characterized by, for example, 5-fold,10-fold, 20-fold or more preferential localization to tumors as comparedto several or many tissue types of non-tumoral tissue, or as comparedto-most or all non-tumoral tissue. Thus, it is understood that, in somecases, a homing molecule homes, in part, to one or more normal organs inaddition to homing to the target tissue. Selective homing can also bereferred to as targeting. The molecules, proteins, cells, tissues, etc.that are targeted by homing molecules can be referred to as targetedmolecules, proteins, cells, tissues, etc.

In some embodiments, one or more of the homing molecules can comprise,consist essentially of, and/or consist of the amino acid sequenceKRGARST (SEQ ID NO: 1) or a conservative derivative thereof, the aminoacid sequence AKRGARSTA (SEQ ID NO: 2) or a conservative derivativethereof, and/or the amino acid sequence CKRGARSTC (SEQ ID NO: 3) or aconservative derivative thereof, or any combination thereof. In someembodiments, one or more of the homing molecule can comprise, consistessentially of, and/or consist of the amino acid sequence KRGARST (SEQID NO: 1) or a conservative variant thereof, and/or the amino acidsequence AKRGARSTA (SEQ ID NO: 2) or a conservative derivative thereof,or any combination thereof. In some embodiments, one or more of thehoming molecules can comprise, consist essentially of, and/or consist ofthe amino acid sequence CKRGARSTC (SEQ ID NO: 3) or a conservativederivative thereof, or any combination thereof.

The composition can comprise a sufficient number and composition ofhoming molecules (modified or not) such that the composition homes tothe target and effectively delivers the cargo molecules. In one example,sufficiency of the number and composition of modified and/or unmodifiedhoming molecules can be determined by assessing cargo delivery and/ortherapeutic effect on the target.

Many homing molecules and homing peptides home to the vasculature of thetarget tissue. However, for the sake of convenience homing is referredto in some places herein as homing to the tissue associated with thevasculature to which the homing molecule or homing peptide may actuallyhome. Thus, for example, a homing molecule that homes to tumorvasculature can be referred to herein as homing to tumor tissue or totumor cells. By including or associating a homing molecule or homingpeptide with, for example, a protein, peptide, amino acid sequence,cargo molecules, or CendR element the protein, peptide, amino acidsequence, cargo molecules, or CendR element can be targeted or can hometo the target of the homing molecule or homing peptide. In this way, theprotein, peptide, amino acid sequence, cargo molecules, or CendR elementcan be said to home to the target of the homing molecule or homingpeptide. For convenience and unless otherwise indicated, reference tohoming of a protein, peptide, amino acid sequence, cargo molecules,CendR element, etc. is intended to indicate that the protein, peptide,amino acid sequence, cargo molecules, CendR element, etc. includes or isassociated with an appropriate homing molecule or homing peptide.

The homing molecule can selectively home to a tumor. The homing moleculecan selectively home to tumor vasculature. The homing molecule canselectively home to one or more particular types of tumor. The homingmolecule can selectively home to the vasculature of one or moreparticular types of tumor. The homing molecule can selectively home toone or more particular stages of a tumor or cancer. The homing moleculecan selectively home to the vasculature of one or more particular stagesof a tumor or cancer. The homing molecule can selectively home to one ormore particular stages of one or more particular types of tumor. Thehoming molecule can selectively home to the vasculature of one or moredifferent stages of one or more particular types of tumor.

The composition can selectively home to a tumor. The composition canselectively home to tumor vasculature. The composition can selectivelyhome to one or more particular types of tumor. The composition canselectively home to the vasculature of one or more particular types oftumor. The composition can selectively home to one or more particularstages of a tumor or cancer. The composition can selectively home to thevasculature of one or more particular stages of a tumor or cancer. Thecomposition can selectively home to one or more particular stages of oneor more particular types of tumor. The composition can selectively hometo the vasculature of one or more different stages of one or moreparticular types of tumor.

The cargo molecule can selectively home to a tumor. The cargo moleculecan selectively home to tumor vasculature. The cargo molecule canselectively home to one or more particular types of tumor. The cargomolecule can selectively home to the vasculature of one or moreparticular types of tumor. The cargo molecule can selectively home toone or more particular stages of a tumor or cancer. The cargo moleculecan selectively home to the vasculature of one or more particular stagesof a tumor or cancer. The cargo molecule can selectively home to one ormore particular stages of one or more particular types of tumor.

The cargo molecule can selectively home to the vasculature of one ormore different stages of one or more particular types of tumor.

The surface molecule can selectively home to a tumor. The surfacemolecule can selectively home to tumor vasculature. The surface moleculecan selectively home to one or more particular types of tumor. Thesurface molecule can selectively home to the vasculature of one or moreparticular types of tumor. The surface molecule can selectively home toone or more particular stages of a tumor or cancer. The surface moleculecan selectively home to the vasculature of one or more particular stagesof a tumor or cancer. The surface molecule can selectively home to oneor more particular stages of one or more particular types of tumor. Thesurface molecule can selectively home to the vasculature of one or moredifferent stages of one or more particular types of tumor.

The membrane perturbing molecule can selectively home to a tumor. Themembrane perturbing molecule can selectively home to tumor vasculature.The membrane perturbing molecule can selectively home to one or moreparticular types of tumor. The membrane perturbing molecule canselectively home to the vasculature of one or more particular types oftumor. The membrane perturbing molecule can selectively home to one ormore particular stages of a tumor or cancer. The membrane perturbingmolecule can selectively home to the vasculature of one or moreparticular stages of a tumor or cancer. The membrane perturbing moleculecan selectively home to one or more particular stages of one or moreparticular types of tumor. The membrane perturbing molecule canselectively home to the vasculature of one or more different stages ofone or more particular types of tumor.

The disclosed compositions, surface molecules, amino acid sequences,cargo molecules, proteins or peptides can, for example, home to braincells, brain stem cells, brain tissue, and/or brain vasculature, kidneycells, kidney stem cells, kidney tissue, and/or kidney vasculature, skincells, skin stem cells, skin tissue, and/or skin vasculature, lungcells, lung tissue, and/or lung vasculature, pancreatic cells,pancreatic tissue, and/or pancreatic vasculature, intestinal cells,intestinal tissue, and/or intestinal vasculature, adrenal gland cells,adrenal tissue, and/or adrenal vasculature, retinal cells, retinaltissue, and/or retinal vasculature, liver cells, liver tissue, and/orliver vasculature, prostate cells, prostate tissue, and/or prostatevasculature, endometriosis cells, endometriosis tissue, and/orendometriosis vasculature, ovary cells, ovary tissue, and/or ovaryvasculature, tumor cells, tumors, tumor blood vessels, and/or tumorvasculature, bone cells, bone tissue, and/or bone vasculature, bonemarrow cells, bone marrow tissue, and/or bone marrow vasculature,cartilage cells, cartilage tissue, and/or cartilage vasculature, stemcells, embryonic stem cells, pluripotent stem cells, induced pluripotentstem cells, adult stem cells, hematopoietic stem cells, neural stemcells, mesenchymal stem cells, mammary stem cells, endothelial stemcells, olfactory adult stem cells, neural crest stem cells, cancer stemcells, blood cells, erythrocytes, platelets, leukocytes, granulocytes,neutrophils, eosinphils, basophils, lymphoid cells, lymphocytes,monocytes, wound vasculature, vasculature of injured tissue, vasculatureof inflamed tissue, atherosclerotic plaques, or a combination.

Examples of homing molecules and homing peptides are known. Examplesinclude brain homing peptides such as CNSRLHLRC (SEQ ID NO: 46),CENWWGDVC (SEQ ID NO: 47), WRCVLREGPAGGCAWFNRHRL (SEQ ID NO: 48),CLSSRLDAC (SEQ ID NO: 49), CVLRGGRC (SEQ ID NO: 50), CNSRLQLRC (SEQ IDNO: 51), CGVRLGC (SEQ ID NO: 52), CKDWGRIC (SEQ ID NO: 53), CLDWGRIC(SEQ ID NO: 54), CTRITESC (SEQ ID NO: 55), CETLPAC (SEQ ID NO: 56),CRTGTLFC (SEQ ID NO: 57), CGRSLDAC (SEQ ID NO: 58), CRHWFDVVC (SEQ IDNO: 59), CANAQSHC (SEQ ID NO: 60), CGNPSYRC (SEQ ID NO: 61),YPCGGEAVAGVSSVRTMCSE (SEQ ID NO: 62), LNCDYQGTNPATSVSVPCTV (SEQ ID NO:63); kidney homing peptides such as CLPVASC (SEQ ID NO: 64), CGAREMC(SEQ ID NO: 65), CKGRSSAC (SEQ ID NO: 66), CWARAQGC (SEQ ID NO: 67),CLGRSSVC (SEQ ID NO: 68), CTSPGGSC (SEQ ID NO: 69), CMGRWRLC (SEQ ID NO:70), CVGECGGC (SEQ ID NO: 71), CVAWLNC (SEQ ID NO: 72), CRRFQDC (SEQ IDNO: 73), CLMGVHC (SEQ ID NO: 74), CKLLSGVC (SEQ ID NO: 75), CFVGHDLC(SEQ ID NO: 76), CRCLNVC (SEQ ID NO: 77), CKLMGEC (SEQ ID NO: 78); skinhoming peptides such as CARSKNKDC (SEQ ID NO: 79), CRKDKC (SEQ ID NO:80), CVALCREACGEGC (SEQ ID NO: 81), CSSGCSKNCLEMC (SEQ ID NO: 82),CIGEVEVC (SEQ ID NO: 83), CKWSRLHSC (SEQ ID NO: 84), CWRGDRKIC (SEQ IDNO: 85), CERVVGSSC (SEQ ID NO: 86),CLAKENVVC (SEQ ID NO: 87); lunghoming peptides such as CGFECVRQCPERC (SEQ ID NO: 88), CGFELETC (SEQ IDNO: 89), CTLRDRNC (SEQ ID NO: 90), CIGEVEVC (SEQ ID NO: 83), CTLRDRNC(SEQ ID NO: 90), CGKRYRNC (SEQ ID NO: 91), CLRPYLNC (SEQ ID NO: 92),CTVNEAYKTRMC (SEQ ID NO: 93), CRLRSYGTLSLC (SEQ ID NO: 94), CRPWHNQAHTEC(SEQ ID NO: 95); pancreas homing peptides such as SWCEPGWCR (SEQ ID NO:96), CKAAKNK (SEQ ID NO: 97), CKGAKAR (SEQ ID NO: 98), VGVGEWSV (SEQ IDNO: 99); intestine homing peptides such as YSGKWGW (SEQ ID NO: 100);uterus homing peptides such as GLSGGRS (SEQ ID NO: 101); adrenal glandhoming peptides such as LMLPRAD (SEQ ID NO: 102), LPRYLLS (SEQ ID NO:103); retina homing peptides such as CSCFRDVCC (SEQ ID NO: 104),CRDVVSVIC (SEQ ID NO: 105); gut homing peptides such as YSGKWGK (SEQ IDNO: 106), GISALVLS (SEQ ID NO: 107), SRRQPLS (SEQ ID NO: 108), MSPQLAT(SEQ ID NO: 109), MRRDEQR (SEQ ID NO: 110), QVRRVPE (SEQ ID NO: 111),VRRGSPQ (SEQ ID NO: 112), GGRGSWE (SEQ ID NO: 113), FRVRGSP (SEQ ID NO:114), RVRGPER (SEQ ID NO: 115); liver homing peptides such as VKSVCRT(SEQ ID NO: 116), WRQNMPL (SEQ ID NO: 117), SRRFVGG (SEQ ID NO: 118),ALERRSL (SEQ ID NO: 119), ARRGWTL (SEQ ID NO: 120); prostate homingpeptides such as SMSIARL (SEQ ID NO: 121), VSFLEYR (SEQ ID NO: 122),RGRWLAL (SEQ ID NO: 123); ovary homing peptides such as EVRSRLS (SEQ IDNO: 124), VRARLMS (SEQ ID NO: 125), RVGLVAR (SEQ ID NO: 126), RVRLVNL(SEQ ID NO: 127); clot binding/homing peptide such as CREKA (SEQ ID NO:128), CGLIIQKNEC (CLT1; SEQ ID NO: 129), CNAGESSKNC (CLT2; SEQ ID NO:130); heart homing peptides such as CRPPR (SEQ ID NO: 131), CGRKSKTVC(SEQ ID NO: 132), CARPAR (SEQ ID NO: 133), CPKRPR (SEQ ID NO: 134),CKRAVR (SEQ ID NO: 135), CRNSWKPNC (SEQ ID NO: 136), RGSSS (SEQ ID NO:137), CRSTRANPC (SEQ ID NO: 138), CPKTRRVPC (SEQ ID NO: 139), CSGMARTKC(SEQ ID NO: 140), GGGVFWQ (SEQ ID NO: 141), HGRVRPH (SEQ ID NO: 142),VVLVTSS (SEQ ID NO: 143), CLHRGNSC (SEQ ID NO: 144), CRSWNKADNRSC (SEQID NO: 145), CGRKSKTVC (SEQ ID NO: 132), CKRAVR (SEQ ID NO: 135),CRNSWKPNC (SEQ ID NO: 136), CPKTRRVPC (SEQ ID NO: 139), CSGMARTKC (SEQID NO: 140), CARPAR (SEQ ID NO: 133), CPKRPR (SEQ ID NO: 134); tumorblood vessel homing peptide such as CNGRC (SEQ ID NO: 146) and otherpeptides with the NGR motif (U.S. Pat. Nos. 6,177,542 and 6,576,239;U.S. patent Application Publication No. 2009/0257951); RGD peptides, andRGR peptides. Other homing peptides include CSRPRRSEC (SEQ ID NO: 147),CSRPRRSVC (SEQ ID NO: 148), and CSRPRRSWC (SEQ ID NO: 149; Hoffman etal., 2003), F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK; SEQ ID NO: 150),PQRRSARLSA (SEQ ID NO: 151), and PKRRSARLSA (SEQ ID NO: 152; U.S. Pat.No. 7,544,767).

Homing molecules can also be defined by their targets. For example,numerous antigens and proteins are known that can be useful fortargeting. Any molecule that can bind, selectively bind, home,selectively, target, selectively target, etc. such target molecules canbe used as a homing molecule. For example, antibodies, nucleic acidaptamers, and compounds that can bind to target molecules can be used ashoming molecules. Examples of useful target molecules for homingmolecules include αv integrins, αvβ3 integrin, αvβ5 integrin, α5β1integrin, aminopeptidase N, tumor endothelial markers (TEMs),endosialin, p32, gC1q receptor, annexin-1, nucleolin, fibronectin ED-B,fibrin-fibronectin complexes, interleukin-11 receptor α, andprotease-cleaved collagen IV. These and other examples are described andreferred to in Ruoslahti et al., 2010, which is hereby incorporated byreference in its entirety and specifically for its description of andreferences to target molecules.

The composition can comprise any number of homing molecules. By way ofexample, the composition can comprise at least 1, 5, 10, 15, 20, 25, 50,75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 625, 750,775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000,75,000, or 100,000, or more homing molecules. The composition can alsocomprise any number in between those numbers listed above.

Homing molecules can be associated with and arranged in the compositionsin a variety of configurations. In some embodiments, homing moleculescan be associated with, conjugated to, and/or covalently coupled to aplurality of surface molecules. In some embodiments, homing moleculescan be associated with, conjugated to, and/or covalently coupled to aplurality of cargo molecules. In some embodiments, homing molecules canbe associated with, conjugated to, and/or covalently coupled to aplurality of cargo molecules, wherein the cargo molecules can beassociated with, conjugated to, and/or covalently coupled to a pluralityof surface molecules. Combinations of these combinations can also beused.

II.D.2.a. Tumor-Homing Molecules

The disclosed homing molecules can be tumor-homing compounds.Tumor-homing compounds are compounds that selectively home to tumors andtumor-associated tissue. Many compounds that target, bind to, and/orhome to tumors are known, most of which can be used as tumor-homingcompounds in the disclosed compositions. Tumor-homing compounds can eachbe independently selected from any known tumor-homing compounds.

Tumor-homing compounds can comprise, consist essentially of, consist ofthe amino acid sequence KRGARST (SEQ ID NO: 1), the amino acid sequenceAKRGARSTA (SEQ ID NO: 2), or a conservative derivative thereof, theamino acid sequence CKRGARSTC (SEQ ID NO: 3) or a conservativederivative thereof, or any combination thereof, includingpeptidomimetics thereof. In some embodiments, one or more of the homingmolecules can comprise, consist essentially of, and/or consist of theamino acid sequence KRGARST (SEQ ID NO: 1), AKRGARSTA (SEQ ID NO: 2),and/or CKRGARSTC (SEQ ID NO: 3).

Useful peptides for tumor targeting include, for example, thetumor-homing CendR peptide iRGD, LyP-1, a peptide that contains aputative CendR element and has tumor-penetrating properties, and RGRpeptides. The LyP-1 peptide has a unique target within tumors; itpreferentially accumulates in the hypoxic/low nutrient areas of tumors(Laakkonen et al., 2002; 2004; Karmali et al., 2009). CRGRRST (RGR; SEQID NO: 153; Joyce et al., 2003) is a peptide that has been successfullyused in targeting a cytokine antibody combination into tumors (Hamzah etal., 2008). This peptide is linear, which simplifies the synthesis. LikeLyP-1, RGR is at least to some extent tumor type-specific (Joyce et al.,2003), but the tumor types recognized by the two peptides seem to bepartially different, which may be an advantage in testing combinationswith the pan-tumor iRGD.

Because tumors can include clot-related proteins, some clot-binding andclot-homing compounds can also be tumor-homing compounds. Suchtumor-homing clot-binding compounds can be used as tumor-homingcompounds as described herein. Tumor-homing compounds can each beindependently selected from, for example, an amino acid segmentcomprising the amino acid sequence REK, an amino acid segment comprisingthe amino acid sequence CAR (such as CARSKNKDC; SEQ ID NO: 79), an aminoacid segment comprising the amino acid sequence CRK (such as CRKDKC; SEQID NO: 80), a fibrin-binding peptide, a peptide that binds clots and notfibrin (such as CGLIIQKNEC (CLT1; SEQ ID NO: 129) and CNAGESSKNC (CLT2;SEQ ID NO: 130)), a clot-binding antibody, and a clot-binding smallorganic molecule. A plurality of the clot-binding compounds can eachindependently comprise an amino acid segment comprising the amino acidsequence REK. Such peptides are also described in U.S. PatentApplication Publication No. 2008/0305101, which is hereby incorporatedby reference for its description of such peptides. Peptides comprisingamino acid sequences CAR or CRK are also described in U.S. PatentApplication Publication No. 2009/0036349, which is hereby incorporatedby reference for its description of such peptides.

LyP-1 are homing molecules that selectively home to tumor lymphaticvasculature, for example, the lymphatic vasculature of breast cancertumors and osteosarcomas, in preference to normal lymphatic vasculature.LyP-1 can selectively home, for example, to the lymphatic vasculature ofsquamous carcinomas. The core LyP-1 peptide has an amino acid sequenceCGNKRTRGC (SEQ ID NO: 7). LyP-1 peptides are described in U.S. PatentApplication Nos. 2004/0087499, 2007/0219134, and 2008/0014143, which arehereby incorporated by reference in their entirety, an specifically fortheir description of such peptides.

The clot-binding compound can also comprise a fibrin-binding peptide(FBP). Examples of fibrin-binding peptides are known in the art (VanRooijen & Sanders, 1994; Moghimi et al., 2001; U.S. Pat. No. 5,792,742,all herein incorporated by reference in their entirety for theirteaching concerning fibrin binding peptides).

Clot-binding peptides can also bind to proteins other than fibrin.Example include peptides that bind to fibronectin that has becomeincorporated into a clot (Pilch et al., 2006, hereby incorporated in itsentirety for its teaching concerning clot-binding peptides). Examples ofclot-binding peptides include, but is not limited to, CGLIIQKNEC (CLT1;SEQ ID NO: 129) and CNAGESSKNC (CLT2; SEQ ID NO: 130). The amino acidsegments can also be independently selected from amino acid segmentscomprising the amino acid sequence CLT 1 or CLT2 or a conservativevariant thereof, amino acid segments comprising the amino acid sequenceCLT1 or CLT2, or amino acid segments consisting of the amino acidsequence CLT1 or CLT2. The amino acid segments can each independentlycomprise the amino acid sequence CLT1 or CLT2 or a conservative variantthereof. The amino acid segments can also each independently comprisethe amino acid sequence CLT1 or CLT2. The amino acid segment can alsoconsist of the amino acid sequence CLT1 or CLT2.

The amino acid segments can also each independently comprise the aminoacid sequence CARSKNKDC (SEQ ID NO: 79), and the amino acid sequence CRK(such as CRKDKC; SEQ ID NO: 80). Peptides comprising amino acidsequences CAR or CRK are also described in U.S. Patent ApplicationPublication No. 2009/0036349, which is hereby incorporated by referencefor its description of such peptides.

The composition can comprise any number of tumor-homing compounds. Byway of example, the composition can comprise at least 1, 5, 10, 15, 20,25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 625,750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000,50,000, 75,000, or 100,000, or more tumor-homing compounds. Thecomposition can also comprise any number in between those numbers listedabove.

Table 3 shows examples of tumor-homing CendR peptides.

TABLE 3 Examples of Tumor-Homing Peptides with CendR ElementsPeptide Sequence Reference(s) CRKDKC (SEQ ID NO: 80)Jarvinen et al., 2007 CGNKRTRGC (SEQ ID NO: 7) Laakkonen et al., 2002AKVKDEPQRRSARLSAKPAPPKPEPK Christian et al., 2003;PKKAPAKK (SEQ ID NO: 154) U.S. Pat. No. 7,544,767CSRPRRSEC (SEQ ID NO: 147) Hoffman et al., 2003CSRPRRSVC (SEQ ID NO: 148) CSRPRRSWC (SEQ ID NO: 149)CNRRTKAGC (SEQ ID NO: 155) Zhang et al., 2006 CRGRRST (SEQ ID NO: 153)Joyce et al., 2003 CRSRKG (SEQ ID NO: 156) CKAAKNK (SEQ ID NO: 97)CKGAKAR (SEQ ID NO: 98) PQRRSARLSA Porkka et al., 2002; (SEQ ID NO: 157)U.S. Pat. No. 7,544,767 PKRRSARLSA U.S. Pat. No. 7,544,767(SEQ ID NO: 158) CRGDKGPDC (SEQ ID NO: 159) iRGD, Sugahara et al., 2009;Sugahara et al., 2010; U.S. Pat. No. 8267,621

Tumor-homing compounds can also be modified. Any of the modificationsdescribed herein for homing molecules can be used with the disclosedtumor-homing compounds.

II.D. 2.b. Modified Homing Molecules

The disclosed homing molecules can include modified forms of homingmolecules. The horning molecules can have any useful modification. Forexample, some modifications can stabilize the homing molecule. Forexample, the disclosed homing molecules include methylated homingmolecules. Methylated homing molecules are particularly useful when thehoming molecule includes a protein, peptide, or amino acid segment. Forexample, a homing molecule can be a modified homing molecule, where, forexample, the modified homing molecule includes a modified amino acidsegment or amino acid sequence. For example, a modified homing moleculecan be a methylated homing molecule, where, for example, the methylatedhoming molecule includes a methylated amino acid segment or amino acidsequence. Other modifications can be used, either alone or incombination. Where the homing molecule is, or includes, a protein,peptide, amino acid segment and/or amino acid sequences, themodification can be to the protein, peptide, amino acid segment, aminoacid sequences, and/or any amino acids in the protein, peptide, aminoacid segment, and/or amino acid sequences. Amino acid and peptidemodifications are known to those of skill in the art, some of which aredescribed below and elsewhere herein. Methylation is a particularlyuseful modification for the disclosed homing molecules. Using modifiedforms of homing molecules can increase the effectiveness of the homingand targeting, which can increase the effect on the target.

A plurality of modified and/or unmodified homing molecules can each beindependently selected from, for example, an amino acid segmentcomprising, consisting essentially of, and/or consisting of a modifiedor unmodified form of the amino acid sequence of a homing peptide, anamino acid segment comprising, consisting essentially of, and/orconsisting of a modified or unmodified form of the amino acid sequenceKRGARST (SEQ ID NO: 1), an amino acid segment comprising, consistingessentially of, and/or consisting of a modified or unmodified form ofthe amino acid sequence AKRGARSTA (SEQ ID NO: 2), and an amino acidsegment comprising, consisting essentially of, and/or consisting of amodified or unmodified form of the amino acid sequence CKRGARSTC (SEQ IDNO: 3). A plurality of the homing molecules can each independentlycomprise an amino acid segment comprising a modified or unmodified formof the amino acid sequence of a homing peptide.

The composition can comprise any number of modified and/or unmodifiedhoming molecules. By way of example, the composition can comprise atleast 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, 625, 750, 775, 800, 825, 850, 875, 900, 925, 950,975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000,7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000,35,000, 40,000, 45,000, 50,000, 75,000, or 100,000, or more modifiedand/or unmodified homing molecules. The composition can also compriseany number in between those numbers listed above.

As used herein, a “methylated derivative” of a protein, peptide, aminoacid segment, amino acid sequence, etc. refers to a form of the protein,peptide, amino acid segment, amino acid sequence, etc. that ismethylated. Unless the context indicates otherwise, reference to amethylated derivative of a protein, peptide, amino acid segment, aminoacid sequence, etc. does not include any modification to the baseprotein, peptide, amino acid segment, amino acid sequence, etc. otherthan methylation. Methylated derivatives can also have othermodifications, but such modifications generally will be noted. Forexample, conservative variants of an amino acid sequence would includeconservative amino acid substitutions of the base amino acid sequence.Thus, reference to, for example, a “methylated derivative” of a specificamino acid sequence “and conservative variants thereof” would includemethylated forms of the specific amino acid sequence and methylatedforms of the conservative variants of the specific amino acid sequence,but not any other modifications of derivations. As another example,reference to a methylated derivative of an amino acid segment thatincludes amino acid substitutions would include methylated forms of theamino acid sequence of the amino acid segment and methylated forms ofthe amino acid sequence of the amino acid segment include amino acidsubstitutions,

II.D.3. Cargo Molecules

The disclosed compositions include one or more cargo molecules.Generally, the disclosed compositions can include a plurality of cargomolecules. The disclosed compositions can include a single type of cargomolecule or a plurality of different types of cargo molecules. Thus, forexample, the disclosed compositions can include a plurality of differenttypes of cargo molecules where a plurality of one or more of thedifferent types of cargo molecules can be present.

Cargo molecules can be any compound, molecule, conjugate, composition,etc. that is desired to be delivered using the disclosed compositions.For example, the cargo molecules can be therapeutic agents, detectableagents, or a combination. For example, the cargo molecules can bemembrane perturbing molecules, pro-apoptotic molecules, pore-generatingmolecules, antimicrobial molecules, mitochondria-affecting molecules,mitochondria-targeted molecules, or a combination. Examples of someuseful cargo molecules are described below and elsewhere herein.

Cargo molecules can be associated with and arranged in the compositionsin a variety of configurations. In some embodiments, cargo molecules canbe associated with, conjugated to, and/or covalently coupled to aplurality of surface molecules. In some embodiments, cargo molecules canbe associated with, conjugated to, and/or covalently coupled to aplurality of homing molecules. In some embodiments, cargo molecules canbe associated with, conjugated to, and/or covalently coupled to aplurality of horning molecules, wherein the homing molecules can beassociated with, conjugated to, and/or covalently coupled to a pluralityof surface molecules. Combinations of these combinations can also beused.

II.D.4.a. Membrane Perturbing Molecules

Useful forms of cargo molecules include membrane perturbing molecules.Membrane perturbing molecules include molecules that can disruptmembranes, which can form pores in membranes, which can make membranesleaky, that can be targeted to or affect intracellular membranes ororganelles, such mitochondria or lysosomes. Some forms of membraneperturbing molecules can be pro-apoptotic while others can benon-apoptotic. Some forms of membrane perturbing molecules can bepro-apoptotic for only some types of cells.

In some embodiments, one or more of the homing molecules can comprise,consist essentially of, and/or consist of the amino acid sequenceKRGARST (SEQ ID NO: 1), the amino acid sequence AKRGARSTA (SEQ ID NO:2), and/or the amino acid sequence CKRGARSTC (SEQ ID NO: 3). In someembodiments, one or more of the membrane perturbing molecules cancomprise the amino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42) or aconservative variant thereof, (KLAKLAK)₂ (SEQ ID NO: 42) or aconservative variant thereof, (KLAKKLA)₂ (SEQ ID NO: 43) or aconservative variant thereof, (KAAKKAA)₂ (SEQ ID NO: 44) or aconservative variant thereof, (KLGKKLG)₃ (SEQ ID) NO: 45) or aconservative variant thereof or a combination. In some embodiments, oneor more of the membrane perturbing molecules can comprise, consistessentially of, and/or consist of the amino acid sequence _(D)(KLAKLAK)₂(SEQ ID NO: 42), (KLAKLAK)₂ (SEQ ID NO: 42), (KLAKKLA)₂ (SEQ ID NO: 43),(KAAKKAA)₂ (SEQ ID NO: 44), (KLGKKLG)₃ (SEQ ID NO: 45), or acombination. In some embodiments, one or more of the membrane perturbingmolecules can comprise, consist essentially of and/or consist of theamino acid sequence _(D)(KLAKLAK)₂ (SEQ ID NO: 42) or a conservativevariant thereof. In some embodiments, one or more of the membraneperturbing molecules can comprise, consist essentially of, and/orconsist of the amino acid sequence _(D)(KLAKLAK)₂ (SEQ II) NO: 42).Membrane perturbing peptides of this type are described in Ellerby,1999, which is hereby incorporated by reference for its description ofsuch peptides.

A plurality of modified and/or unmodified membrane perturbing moleculescan each be independently selected from, for example, an amino acidsegment comprise, consist essentially of, and/or consist of a modifiedor unmodified form of the amino acid sequence of a homing peptide, anamino acid segment comprise, consist essentially of, and/or consist of amodified or unmodified form of the amino acid sequence _(D)(KLAKLAK)₂(SEQ ID NO: 42), (KLAKLAK)₂ (SEQ ID NO: 42), (KLAKKLA)₂ (SEQ ID NO.:43), (KAAKKAA)₂ (SEQ ID) NO: 44), (KLGKKLG)₃ (SEQ ID) NO: 45), or anycombination thereof. A plurality of the membrane perturbing moleculescan each independently comprise an amino acid segment comprising amodified or unmodified form of the amino acid sequence of a homingpeptide.

The composition can comprise a sufficient number and composition ofmembrane perturbing molecules (modified or not) such that thecomposition has a membrane perturbing effect on the target. In oneexample, sufficiency of the number and composition of modified and/orunmodified membrane perturbing molecules can be determined by assessingmembrane disruption, apoptosis, and/or therapeutic effect on the target.

The composition can comprise any number of modified and/or unmodifiedmembrane perturbing molecules. By way of example, the composition cancomprise at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 625, 750, 775, 800, 825, 850, 875, 900,925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000,6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 75,000, or 100,000, ormore modified and/or unmodified membrane perturbing molecules. Thecomposition can also comprise any number in between those numbers listedabove.

Membrane perturbing molecules can be associated with and arranged in thecompositions in a variety of configurations. In some embodiments,membrane perturbing molecules can be associated with, conjugated to,and/or covalently coupled to a plurality of surface molecules. In someembodiments, membrane perturbing molecules can be associated with,conjugated to, and/or covalently coupled to a plurality of homingmolecules. In some embodiments, membrane perturbing molecules can beassociated with, conjugated to, and/or covalently coupled to a pluralityof homing molecules, wherein the homing molecules can be associatedwith, conjugated to, and/or covalently coupled to a plurality of surfacemolecules. Combinations of these combinations can also be used.

II.D.4.b. Modified Membrane Perturbing Molecules

The disclosed membrane perturbing molecules can include modified formsof membrane perturbing molecules. The membrane perturbing molecules canhave any useful modification. For example, some modifications canstabilize the membrane perturbing molecule. For example, the disclosedmembrane perturbing molecules include methylated membrane perturbingmolecules. Methylated membrane perturbing molecules are particularlyuseful when the membrane perturbing molecule includes a protein,peptide, or amino acid segment. For example, a membrane perturbingmolecule can be a modified membrane perturbing molecule, where, forexample, the modified membrane perturbing molecule includes a modifiedamino acid segment or amino acid sequence. For example, a modifiedmembrane perturbing molecule can be a methylated membrane perturbingmolecule, where, for example, the methylated membrane perturbingmolecule includes a methylated amino acid segment or amino acidsequence. Other modifications can be used, either alone or incombination. Where the membrane perturbing molecule is, or includes, aprotein, peptide, amino acid segment and/or amino acid sequences, themodification can be to the protein, peptide, amino acid segment, aminoacid sequences, and/or any amino acids in the protein, peptide, aminoacid segment, and/or amino acid sequences. Amino acid and peptidemodifications are known to those of skill in the art, some of which aredescribed below and elsewhere herein. Methylation is a particularlyuseful modification for the disclosed membrane perturbing molecules.Using modified forms of membrane perturbing molecules can increase theireffectiveness.

II.E.1. Moieties Generally

Disclosed are compositions useful for directing a moiety to a target.For example, the moiety can be incorporated into a TT1 Peptidecomposition. As used herein, the term “moiety” is used broadly to mean aphysical, chemical, or biological material that generally imparts abiologically useful function to a linked molecule. A moiety can be anynatural or non-natural material including, without limitation, abiological material, such as a cell, phage or other virus; an organicchemical such as a small molecule; a radionuclide; a nucleic acidmolecule or oligonucleotide; a polypeptide; or a peptide. Usefulmoieties include, but are not limited to, therapeutic agents such ascancer chemotherapeutic agents, cytotoxic agents, pro-apoptotic agents,and anti-angiogenic agents; detectable agents and imaging agents; andtags or other insoluble supports. Useful moieties further include,without limitation, phage and other viruses, cells, liposomes, polymericmatrices, non-polymeric matrices or particles such as gold particles,microdevices and nanodevices, and nanoscale semiconductor materials.These and other moieties known in the art can be components of aconjugate.

Thus, in some embodiments the composition can further comprise one ormore moieties. In some embodiments, the moieties can be independentlyselected from the group consisting of an anti-angiogenic agent, apro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxicagent, an anti-inflammatory agent, an anti-arthritic agent, apolypeptide, a nucleic acid molecule, a small molecule, an imagecontrast agent, a fluorophore, fluorescein, rhodamine, a radionuclide,indium-111, technetium-99, carbon-11, and carbon-13. In someembodiments, at least one of the moieties can be a therapeutic agent. Insome embodiments, the therapeutic agent can be iRGD, RGD, Abraxane,paclitaxel, taxol, or a combination. In some embodiments, at least oneof the moieties can be a detectable agent. In some embodiments, thedetectable agent can be FAM.

II.E.2. Therapeutic Agents

In some embodiments, the composition can have a therapeutic effect. Insome embodiments, the composition can reduce tumor growth. In someembodiments, the therapeutic effect can be a slowing in the increase ofor a reduction of tumor burden. In some embodiments, the therapeuticeffect can be a slowing of the increase of or reduction of tumor size.In some embodiments, the subject can have one or more sites targeted,wherein the composition can home to one or more of the sites targeted.In some embodiments, the subject can have a tumor, wherein thecomposition can have a therapeutic effect on the tumor. The moiety canbe a therapeutic agent. As used herein, the term “therapeutic agent”means a molecule which has one or more biological activities in a normalor pathologic tissue. A variety of therapeutic agents can be used as amoiety.

In some embodiments, the therapeutic agent can be a cancerchemotherapeutic agent. As used herein, a “cancer chemotherapeuticagent” is a chemical agent that inhibits the proliferation, growth,life-span or metastatic activity of cancer cells. Such a cancerchemotherapeutic agent can be, without limitation, a taxane such asdocetaxel; an anthracyclin such as doxorubicin; an alkylating agent; avinca alkaloid; an anti-metabolite; a platinum agent such as cisplatinor carboplatin; a steroid such as methotrexate; an antibiotic such asadriamycin; a isofamide; or a selective estrogen receptor modulator; anantibody such as trastuzumab.

Taxanes are chemotherapeutic agents useful in Lyp-1 compositions. Usefultaxanes include, without limitation, docetaxel (Taxotere; AventisPharmaceuticals, Inc.; Parsippany, N.J., United States of America) andpaclitaxel (Taxol; Bristol-Myers Squibb; Princeton, N.J., United Statesof America). See e.g., Chan et al., 1999; Paridaens et al., 2000.

A cancer chemotherapeutic agent useful in a TT1 Peptide composition alsocan be an anthracyclin such as doxorubicin, idarubicin, or daunorubicin.Doxorubicin is a commonly used cancer chemotherapeutic agent and can beuseful, for example, for treating breast cancer (Stewart & Ratain, 1997;Harris et al., 1997). In addition, doxorubicin has anti-angiogenicactivity (Folkman, 1997; Steiner et al., 1992), which can contribute toits effectiveness in treating cancer.

An alkylating agent such as melphalan or chlorambucil also can be auseful cancer chemotherapeutic agent. Similarly, a vinca alkaloid suchas vindesine, vinblastine, or vinorelbine; or an antimetabolite such as5-fluorouracil, 5-fluorouridine, or a derivative thereof can be a usefulcancer chemotherapeutic agent.

A platinum agent also can be a useful cancer chemotherapeutic agent.Such a platinum agent can be, for example, cisplatin or carboplatin asdescribed, for example, in Crown (2001) Sem Oncol 28:28-37. Other usefulcancer chemotherapeutic agents include, without limitation,methotrexate, mitomycin-C, adriamycin, ifosfamide, and ansamycins.

A cancer chemotherapeutic agent useful for treatment of breast cancerand other hormonally-dependent cancers also can be an agent thatantagonizes the effect of estrogen, such as a selective estrogenreceptor modulator or an anti-estrogen. The selective estrogen receptormodulator, tamoxifen, is a cancer chemotherapeutic agent that can beused in a conjugate for treatment of breast cancer (Fisher et al.,1998).

The therapeutic agent can be an antibody such as a humanized monoclonalantibody. As an example, the anti-epidermal growth factor receptor 2(HER2) antibody, trastuzumab (HERCEPTIN®; Genentech, South SanFrancisco, Calif., United States of America) can be a therapeutic agentuseful for treating HER2/neu overexpressing breast cancers (White etal., 2001).

Useful therapeutic agents also can be a cytotoxic agent, which, as usedherein, can be any molecule that directly or indirectly promotes celldeath. Useful cytotoxic agents include, without limitation, smallmolecules, polypeptides, peptides, peptidomimetics, nucleicacid-molecules, cells, and viruses. As non-limiting examples, usefulcytotoxic agents include cytotoxic small molecules such as doxorubicin,docetaxel or trastuzumab; antimicrobial peptides such as those describedfurther below; pro-apoptotic polypeptides such as caspases and toxins,for example, caspase-8; diphtheria toxin A chain, Pseudomonas exotoxinA, cholera toxin, ligand fusion toxins such as DAB(389)EGF, ricinuscommunis toxin (ricin); and cytotoxic cells such as cytotoxic T cells.See e.g., Martin et al., 2000; Kreitman & Pastan, 1997; Allam et al.,1997; Osborne & Coronado-Heinsohn, 1996. One skilled in the artunderstands that these and additional cytotoxic agents described hereinor known in the art can be useful in the disclosed conjugates andmethods.

In some embodiments, a therapeutic agent can be a therapeuticpolypeptide. As used herein, a therapeutic polypeptide can be anypolypeptide with a biologically useful function. Useful therapeuticpolypeptides encompass, without limitation, cytokines, antibodies,cytotoxic polypeptides; pro-apoptotic polypeptides; and anti-angiogenicpolypeptides. As non-limiting examples, useful therapeutic polypeptidescan be a cytokine such as tumor necrosis factor-α (TNF-α), tumornecrosis factor-β (TNF-β), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF),interferon alpha (IFN-α); interferon gamma (IFN-γ), interleukin-1(IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4(IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10(IL-10), interleukin-12 (IL-12), lymphotactin (LTN), or dendritic cellchemokine 1 (DC-CK1); an anti-HER2 antibody or fragment thereof; acytotoxic polypeptide including a toxin or caspase, for example,diphtheria toxin A chain, Pseudomonas exotoxin A, cholera toxin, aligand fusion toxin such as DAB(389)EGF (see U.S. Pat. No. 5,906,820) orricin; or an anti-angiogenic polypeptide such as angiostatin,endostatin, thrombospondin, platelet factor 4; anastellin; or one ofthose described further herein or known in the art (see below). It isunderstood that these and other polypeptides with biological activitycan be a “therapeutic polypeptide.”

A therapeutic agent can also be an anti-angiogenic agent. As usedherein, the term “anti-angiogenic agent” means a molecule that reducesor prevents angiogenesis, which is the growth and development of bloodvessels. A variety of anti-angiogenic agents can be prepared by routinemethods. Such anti-angiogenic agents include, without limitation, smallmolecules; proteins such as dominant negative forms of angiogenicfactors, transcription factors, and antibodies; peptides; and nucleicacid molecules including ribozymes, antisense oligonucleotides, andnucleic acid molecules encoding, for example, dominant negative forms ofangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof. See e.g., Hagedom & Bikfalvi,2000; Kirsch et al., 2000.

Vascular endothelial growth factor (VEGF) has been shown to be importantfor angiogenesis in many types of cancer, including breast cancerangiogenesis in vivo (Borgstrom et al., 1999). The biological effects ofVEGF include stimulation of endothelial cell proliferation, survival,migration, and tube formation, and regulation of vascular permeability.An anti-angiogenic agent can be, for example, an inhibitor orneutralizing antibody that reduces the expression or signaling of VEGFor another angiogenic factor, for example, an anti-VEGF neutralizingmonoclonal antibody (Borgstrom et al., 1999). An anti-angiogenic agentalso can inhibit another angiogenic factor such as a member of thefibroblast growth factor family such as FGF-1 (acidic), FGF-2 (basic),FGF-4, or FGF-5 (Slavin et al., 1995; Folkman & Shing, 1992) or anangiogenic factor such as angiopoietin-1, a factor that signals throughthe endothelial cell-specific Tie2 receptor tyrosine kinase (Davis etal., 1996; Suri et al., 1996), or the receptor of one of theseangiogenic factors. It is understood that a variety of mechanisms canact to inhibit activity of an angiogenic factor including, withoutlimitation, direct inhibition of receptor binding, indirect inhibitionby reducing secretion of the angiogenic factor into the extracellularspace, or inhibition of expression, function or signaling of theangiogenic factor.

A variety of other molecules also can function as anti-angiogenic agentsincluding, without limitation, angiostatin; a kringle peptide ofangiostatin; endostatin; anastellin, heparin-binding fragments offibronectin; modified forms of antithrombin; collagenase inhibitors;basement membrane tumover inhibitors; angiostatic steroids; plateletfactor 4 and fragments and peptides thereof; thrombospondin andfragments and peptides thereof; and doxorubicin (O'Reilly et al., 1994;O'Reilly et al., 1997; Homandberg et al., 1985; Homandberg et al., 1986;O'Reilly et al., 1999). Commercially available anti-angiogenic agentsinclude, for example, angiostatin, endostatin, metastatin and 2ME2(EntreMed; Rockville, Md., United States of America); anti-VEGFantibodies such as Avastin (Genentech; South San Francisco, Calif.,United States of America); and VEGFR-2 inhibitors such as SU5416(3-[(3,5-Dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one),a small molecule inhibitor of VEGFR-2 (SUGEN; South San Francisco,Calif., United States of America) and SU6668(5-[1,2-Dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoicacid; SUGEN), a small molecule inhibitor of VEGFR-2, platelet derivedgrowth factor and fibroblast growth factor I receptor. It is understoodthat these and other anti-angiogenic agents can be prepared by routinemethods and are encompassed by the term “anti-angiogenic agent” as usedherein.

The TT1 Peptide compositions disclosed herein can also be used to siteof inflammation. Moieties useful for this purpose can includetherapeutic agents belonging to several basic groups includinganti-inflammatory agents which prevent inflammation, restenosispreventing drugs which prevent tissue growth, anti-thrombogenic drugswhich inhibit or control formation of thrombus or thrombolytics, andbioactive agents which regulate tissue growth and enhance healing of thetissue. Examples of useful therapeutic agents include but are notlimited to steroids, fibronectin, anti-clotting drugs, anti-plateletfunction drugs, drugs which prevent smooth muscle cell growth on innersurface wall of vessel, heparin, heparin fragments, aspirin, coumadin,tissue plasminogen activator (TPA), urokinase, hirudin, streptokinase,antiproliferatives (methotrexate, cisplatin, fluorouracil, Adriamycin),antioxidants (ascorbic acid, beta carotene, vitamin E), antimetabolites,thromboxane inhibitors, non-steroidal and steroidal anti-inflammatorydrugs, beta and calcium channel blockers, genetic materials includingDNA and RNA fragments, complete expression genes, antibodies,lymphokines, growth factors, prostaglandins, leukotrienes, laminin,elastin, collagen, and integrins.

Useful therapeutic agents also can be antimicrobial peptides. This canbe particularly useful to target a wound or other infected sites. Thus,for example, also disclosed are TT1 Peptide compositions comprising anantimicrobial peptide, where the TT1 Peptide composition is selectivelyinternalized and exhibits a high toxicity to the targeted area. Usefulantimicrobial peptides can have low mammalian cell toxicity when notincorporated into the TT1 Peptide composition. As used herein, the term“antimicrobial peptide” means a naturally occurring or synthetic peptidehaving antimicrobial activity, which is the ability to kill or slow thegrowth of one or more microbes. An antimicrobial peptide can, forexample, kill or slow the growth of one or more strains of bacteriaincluding a Gram-positive or Gram-negative bacteria, or a fungi orprotozoa. Thus, an antimicrobial peptide can have, for example,bacteriostatic or bacteriocidal activity against, for example, one ormore strains of Escherichia coli, Pseudomonas aeruginosa, orStaphylococcus aureus. While not wishing to be bound by the following,an antimicrobial peptide can have biological activity due to the abilityto form ion channels through membrane bilayers as a consequence ofself-aggregation.

An antimicrobial peptide is typically highly basic and can have a linearor cyclic structure. As discussed further below, an antimicrobialpeptide can have an amphipathic α-helical structure (see e.g., U.S. Pat.No. 5,789,542; Javadpour et al., 1996; Blondelle & Houghten, 1992a). Anantimicrobial peptide also can be, for example, a β-strand/sheet-formingpeptide as described in Mancheno et al., 1998.

An antimicrobial peptide can be a naturally occurring or syntheticpeptide. Naturally occurring antimicrobial peptides have been isolatedfrom biological sources such as bacteria, insects, amphibians, andmammals and are thought to represent inducible defense proteins that canprotect the host organism from bacterial infection. Naturally occurringantimicrobial peptides include the gramicidins, magainins, mellitins,defensins, and cecropins (see e.g., Maloy & Kari, 1995; Alvarez-Bravo etal., 1994; Bessalle et al., 1990; Blondelle & Houghten, 1992b). Anantimicrobial peptide also can be an analog of a natural peptide,especially one that retains or enhances amphipathicity (see below).

An antimicrobial peptide incorporated into a TT1 Peptide composition canhave low mammalian cell toxicity linked to a TT1 Peptide. Mammalian celltoxicity readily can be assessed using routine assays. As an example,mammalian cell toxicity can be assayed by lysis of human erythrocytes invitro as described in Javadpour et al., 1996. An antimicrobial peptidehaving low mammalian cell toxicity is not lytic to human erythrocytes orrequires concentrations of in some embodiments greater than 100 μM forlytic activity, and requires concentrations of greater than in someembodiments 200 μM, in some embodiments 300 μM, in some embodiments 500μM, and in some embodiments 1000 μM.

In some embodiments, disclosed are TT1 Peptide compositions in which theantimicrobial peptide portion promotes disruption of mitochondrialmembranes when internalized by eukaryotic cells. In particular, such anantimicrobial peptide preferentially disrupts mitochondrial membranes ascompared to eukaryotic membranes. Mitochondrial membranes, likebacterial membranes but in contrast to eukaryotic plasma membranes, havea high content of negatively charged phospholipids. An antimicrobialpeptide can be assayed for activity in disrupting mitochondrialmembranes using, for example, an assay for mitochondrial swelling oranother assay well known in the art. _(D)(KLAKLAK)₂, for example, is anantimicrobial peptide which induces marked mitochondrial swelling at aconcentration of 10 μM, significantly less than the concentrationrequired to kill eukaryotic cells.

An antimicrobial peptide that induces significant mitochondrial swellingat, for example, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, or less, isconsidered a peptide that promotes disruption of mitochondrialmembranes.

Antimicrobial peptides generally have random coil conformations indilute aqueous solutions, yet high levels of helicity can be induced byhelix-promoting solvents and amphipathic media such as micelles,synthetic bilayers, or cell membranes. α-Helical structures are wellknown in the art, with an ideal α-helix characterized by having 3.6residues per turn and a translation of 1.5 Å per residue (5.4 Å perturn; see Creighton, 1984). In an amphipathic α-helical structure, polarand non-polar amino acid residues are aligned into an amphipathic helix,which is an α-helix in which the hydrophobic amino acid residues arepredominantly on one face, with hydrophilic residues predominantly onthe opposite face when the peptide is viewed along the helical axis.

Antimicrobial peptides of widely varying sequence have been isolated,sharing an amphipathic α-helical structure as a common feature (Saberwalet al., 1994). Analogs of native peptides with amino acid substitutionspredicted to enhance amphipathicity and helicity typically haveincreased antimicrobial activity. In general, analogs with increasedantimicrobial activity also have increased cytotoxicity againstmammalian cells (Maloy et al., 1995).

As used herein in reference to an antimicrobial peptide, the term“amphipathic α-helical structure” refers to an α-helix with ahydrophilic face containing several polar residues at physiological pHand a hydrophobic face containing nonpolar residues. A polar residue canbe, for example, a lysine or arginine residue, while a nonpolar residuecan be, for example, a leucine or alanine residue. An antimicrobialpeptide having an amphipathic.alpha.-helical structure generally has anequivalent number of polar and nonpolar residues within the amphipathicdomain and a sufficient number of basic residues to give the peptide anoverall positive charge at neutral pH (Saberwal et al., 1994). Oneskilled in the art understands that helix-promoting amino acids such asleucine and alanine can be advantageously included in an antimicrobialpeptide (see e.g., Creighton, 1984 supra). Synthetic, antimicrobialpeptides having an amphipathic a-helical structure are known in the art,for example, as described in U.S. Pat. No. 5,789,542 to McLaughlin &Becker.

It is understood by one skilled in the art of medicinal oncology thatthese and other agents are useful therapeutic agents, which can be usedseparately or together in the disclosed compositions and methods. Thus,it is understood that a TT1 Peptide composition can contain one or moreof such therapeutic agents and that additional components can beincluded as part of the composition, if desired. As a non-limitingexample, it can be desirable in some cases to utilize an oligopeptidespacer between a TT1 Peptide and the therapeutic agent (Fitzpatrick &Garnett, 1995).

Other useful agents include thrombolytics, aspirin, anticoagulants,painkillers and tranquilizers, beta-blockers, ace-inhibitors, nitrates,rhythm-stabilizing drugs, and diuretics. Agents that limit damage to theheart work best if given within a few hours of the heart attack.Thrombolytic agents that break up blood clots and enable oxygen-richblood to flow through the blocked artery increase the patient's chanceof survival if given as soon as possible after the heart attack.Thrombolytics given within a few hours after a heart attack are the mosteffective. Injected intravenously, these include anisoylated plasminogenstreptokinase activator complex (APSAC) or anistreplase, recombinanttissue-type plasminogen activator (r-tPA), and streptokinase. Thedisclosed TT1 Peptide compositions can use any of these or similaragents.

Some other examples of useful therapeutic agents include nitrogenmustards, nitrosoureas, ethyleneimine, alkane sulfonates, tetrazine,platinum compounds, pyrimidine analogs, purine analogs, antimetabolites,folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomeraseinhibitors and hormonal agents. Exemplary chemotherapy drugs areActinomycin-D, Alkeran, Ara-C, Anastrozole, Asparaginase, BiCNU,Bicalutamide, Bleomycin, Busulfan, Capecitabine, Carboplatin,Carboplatinum, Carmustine, CCNU, Chlorambucil, Chlomaphazine,Cholophosphamide, Cisplatin, Cladribine, CPT-11, Cyclophosphamide,Cytarabine, Cytosine arabinoside, Cytoxan, Dacarbazine, Dactinomycin,Daunorubicin, Dexrazoxane, Docetaxel, Doxorubicin, DTIC, Epirubicin,Estramustine, Ethyleneimine, Etoposide, Floxuridine, Fludarabine,Fluorouracil, Flutamide, Fotemustine, Gemcitabine, Herceptin,Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan,Lomustine, Mechlorethamine, mechlorethamine oxide hydrochloride,Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane,Mitoxantrone, Novembiehin, Oxaliplatin, Paclitaxel, Pamidronate,Pentostatin, Phenesterine, Plicamycin, Prednimustine, Procarbazine,Rituximab, Steroids, Streptozocin, STI-571, Streptozocin, Tamoxifen,Temozolomide, Teniposide, Tetrazine, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulphan, Trimetrexate, Trofosfamide, Vinblastine,Vincristine, Vindesine, Vinorelbine, VP-16, and Xeloda. Alkylatingagents such as Thiotepa and; alkyl sulfonates such as Busulfan,Improsulfan and Piposulfan; aziridines such as Benzodopa, Carboquone,Meturedopa, and Uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitroureas suchas Cannustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, andRanimustine; antibiotics such as Aclacinomysins, Actinomycin,Authramycin, Azaserine, Bleomycins, Cactinomycin, Calicheamicin,Carabicin, Caminomycin, Carzinophilin, Chromoinycins, Dactinomycin,Daunorubicin, Detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin,Epirubicin, Esorubicin, Idambicin, Marcellomycin, Mitomycins,mycophenolic acid, Nogalamycin, Olivomycins, Peplomycin, Potfiromycin,Puromycin, Quelamycin, Rodorubicin, Streptonigrin, Streptozocin,Tubercidin, Ubenimex, Zinostatin, and Zorubicin; anti-metabolites suchas Methotrexate and 5-fluorouracil (5-FU); folic acid analogues such asDenopterin, Methotrexate, Pteropterin, and Trimetrexate; purine analogssuch as Fludarabine, 6-mercaptopurine, Thiamiprine, and Thioguanine;pyrimidine analogs such as Ancitabine, Azacitidine, 6-azauridine,Carmofur, Cytarabine, Dideoxyuridine, Doxifluridine, Enocitabine,Floxuridine, and 5-FU; androgens such as Calusterone, DromostanolonePropionate, Epitiostanol, Rnepitiostane, and Testolactone; anti-adrenalssuch as aminoglutethimide, Mitotane, and Trilostane; folic acidreplenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; Amsacrine; Bestrabucil; Bisantrene;Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfornithine;elliptinium acetate; Etoglucid; gallium nitrate; hydroxyurea; Lentinan;Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine;Pentostatin; Phenamet; Pirarubicin; podophyllinic acid;2-ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofrran;Spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; Urethan; Vindesine; Dacarbazine;Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine;Arabinoside (“Ara-C”); cyclophosphamide; thiotEPa; taxoids, e.g.,Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) andDoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); Gemcitabine;6-thioguanine; Mercaptopurine; Methotrexate; platinum analogs such asCisplatin and Carboplatin; Vinblastine; platinum; etoposide (VP-16);Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine;Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda;Ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; Esperamicins;Capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included are anti-hormonal agentsthat act to regulate or inhibit hormone action on tumors such asanti-estrogens including for example Tamoxifen, Raloxifene, aromataseinhibiting 4(5)-imidazoles, 4 Hydroxytamoxifen, Trioxifene, Keoxifene,Onapristone, And Toremifene (Fareston); and anti-androgens such asFlutamide, Nilutamide, Bicalutamide, Leuprolide, and Goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Useful cargo molecules include, for example, doxorubicin,Herceptin, and liposomal doxorubicin.

The cargo molecules can also comprise a boron containing compound. Boroncontaining compounds have received increasing attention as therapeuticagents over the past few years as technology in organic synthesis hasexpanded to include this atom (Groziak, 2001). The most notable boroncontaining therapeutic is the boronic acid bortezomib which was recentlylaunched for the treatment of multiple myeloma. This breakthroughdemonstrates the feasibility of using boron containing compounds aspharmaceutical agents. Boron containing compounds have been shown tohave various biological activities including herbicides (German PatentApplication Publication No. DE 1016978 19571003), boron neutron capturetherapy (Yamamoto, 1991), serine protease inhibition (Simpelkamp &Jones, 1992); Weinand et al., 1999), acetylcholinesterase inhibition(Koehler & Hess, 1974) and as antibacterial agents (Bailey et al.,1980). The boron containing compounds with antibacterial activity can besub-divided into two main classes, the diazaborinines, which have beenknown since the 1960's, and dithienylborinic acid complexes. This latterclass has been expanded to include many different diarylborinic acidcomplexes with potent antibacterial activity (PCT International PatentApplication Publication No. WO 2002/044184).

II.E.3. Detectable Agents

A moiety in the disclosed TT1 Peptide compositions can also be adetectable agent. A variety of detectable agents are useful in thedisclosed methods. As used herein, the term “detectable agent” refers toany molecule which can be detected. Useful detectable agents includecompounds and molecules that can be administered in vivo andsubsequently detected. Detectable agents useful in the disclosedcompositions and methods include yet are not limited to radiolabels andfluorescent molecules. The detectable agent can be, for example, anymolecule that facilitates detection, either directly or indirectly,preferably by a non-invasive and/or in vivo visualization technique. Forexample, a detectable agent can be detectable by any known imagingtechniques, including, for example, a radiological technique. Detectableagents can include, for example, a contrasting agent, e.g., where thecontrasting agent is ionic or non-ionic. In some embodiments, forinstance, the detectable agent comprises a tantalum compound and/or abarium compound, e.g., barium sulfate. In some embodiments, thedetectable agent comprises iodine, such as radioactive iodine. In someembodiments, for instance, the detectable agent comprises an organiciodo acid, such as iodo carboxylic acid, triiodophenol, iodoform, and/ortetraiodoethylene. In some embodiments, the detectable agent comprises anon-radioactive detectable agent, e.g., a non-radioactive isotope. Forexample, Gd can be used as a non-radioactive detectable agent in certainembodiments.

Other examples of detectable agents include molecules which emit or canbe caused to emit detectable radiation (e.g., fluorescence excitation,radioactive decay, spin resonance excitation, etc.), molecules whichaffect local electromagnetic fields (e.g., magnetic, ferromagnetic,ferromagnetic, paramagnetic, and/or superparamagnetic species),molecules which absorb or scatter radiation energy (e.g., chromophoresand/or fluorophores), quantum dots, heavy elements and/or compoundsthereof. See e.g., detectable agents described in U.S. PatentApplication Publication No. 2004/0009122. Other examples of detectableagents include a proton-emitting molecules, a radiopaque molecules,and/or a radioactive molecules, such as a radionuclide like Tc-99mand/or Xe-13. Such molecules can be used as a radiopharmaceutical. Instill other embodiments, the disclosed compositions can comprise one ormore different types of detectable agents, including any combination ofthe detectable agents disclosed herein.

Useful fluorescent moieties include, but are not limited to fluoresceinisothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®,CASCADE BLUE®, OREGON GREEN®, pyrene, lissamine, xanthenes, acridines,oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such asQUANTUM DYE™, fluorescent energy transfer dyes, such as thiazoleorange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. Non-limiting examples of other fluorescent labels include3-Hydroxypyrene 5,8,10-Trisulfonic acid, 5-Hydroxytryptamine (5-HT),Acid Fuchsin, Alizarin Complexone, Alizarin Red, Allophycocyanin,Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, AstrazonOrange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF,Berberine Sulfate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV,BODIPY® F1, Brilliant Sulfoflavin FF, Calcien Blue, Calcium Green,Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution,Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.18, CY5.1 8, CY7, Dans (1-Dimethyl-aminonaphthalene-5-sulfonate), Dansa(Diamino Naphtyl Sulfonic Acid), Dansyl NH—CH3, Diamino PhenylOxydiazole (DAO), Dimethylamino-5-Sulfonic acid, DipyrrometheneboronDifluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC,Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl BrilliantYellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid,Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (MethylGreen Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), PhorwiteAR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black,Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron BrilliantRed 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange,Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulfonic acid),Stilbene, Snarf 1, sulfoRhodamine B Can C, Sulfo Rhodamine G Extra,Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue, Ultralite, UranineB, Uvitex SFC, Xylene Orange, and XRITC.

Particularly useful fluorescent labels include fluorescein(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. The absorption and emission maxima, respectively, for thesefluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm;588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm;778 nm), thus allowing their simultaneous detection. Other examples offluorescein dyes include 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE),2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein(NED), and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).Fluorescent labels can be obtained from a variety of commercial sources,including Amersham Pharmacia Biotech, Piscataway, N.J. United States ofAmerica; Molecular Probes, Eugene, Oreg., United States of America; andResearch Organics, Cleveland, Ohio, United States of America.Fluorescent probes and there use are also described in Haugland, 2002.

Further examples of radioactive detectable agents include gamma emitters(e.g., the gamma emitters In-111, I-125, and I-131; Rhenium-186 and 188;and Br-77; see e.g., Thakur et al., 1976; Powers et al., 1982; U.S. Pat.No. 5,011,686); positron emitters, such as but not limited to Cu-64,C-11, and O-15; as well as Co-57, Cu-67, Ga-67, Ga-68, Ru-97, Tc-99m,In-113m, Hg-197, Au-198, and Pb-203. Other radioactive detectable agentscan include, for example tritium, C-14 and/or thallium, as well asRh-105, 1-123, Nd-147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, and/orT1-201.

In some embodiments, Technitium-99m (Tc-99m) is employed as has beendescribed in other applications (see e.g., U.S. Pat. Nos. 4,418,052 and5,024,829). Tc-99m is a gamma emitter with single photon energy of 140keV and a half-life of about 6 hours, and can readily be obtained from aMo-99/Tc-99 generator.

In some embodiments, compositions comprising a radioactive detectableagent can be prepared by coupling a targeting moiety with radioisotopessuitable for detection. Coupling can occur via a chelating agent such asdiethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and/ormetallothionein, any of which can be covalently attached to thetargeting moiety. In some embodiments, an aqueous mixture of Tc-99m, areducing agent, and a water-soluble ligand can be prepared and thenallowed to react with a disclosed targeting moiety. Such methods areknown in the art (see e.g., PCT International Patent ApplicationPublication No. WO 99/64446). In some embodiments, compositionscomprising radioactive iodine can be prepared using an exchangereaction. For example, exchange of hot iodine for cold iodine is wellknown in the art. Alternatively, a radioiodine-labeled compound can beprepared from the corresponding bromo compound via a tributylstannylintermediate.

Magnetic detectable agents include paramagnetic contrasting agents, forexample gadolinium diethylenetriaminepentaacetic acid, which can beused, for example, in magnetic resonance imaging (MRI; see e.g., De Rooset al., 1991). Exemplary, non-limiting embodiments of the presentlydisclosed subject matter can use as the detectable agent paramagneticatoms that are divalent or trivalent ions of elements with an atomicnumber 21, 22, 23, 24, 25, 26, 27, 28, 29, 42, 44, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, or 70. Suitable ions include, but are notlimited to, chromium(III), manganese(II), iron(II), iron(III),cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III),samarium(III) and ytterbium(III), as well as gadolinium(III),terbium(III), dysoprosium(III), holmium(III), and erbium(III). In someembodiments, a magnetic detectable agent employs atoms with strongmagnetic moments, e.g., gadolinium(III).

In some embodiments, compositions comprising magnetic detectable agentscan be prepared by coupling a targeting moiety with a paramagnetic atom.For example, the metal oxide or a metal salt, such as a nitrate,chloride or sulfate salt, of a suitable paramagnetic atom can bedissolved or suspended in a water/alcohol medium, such as methyl, ethyl,and/or isopropyl alcohol. The mixture can be added to a solution of anequimolar amount of the targeting moiety in a similar water/alcoholmedium and stirred. The mixture can be heated moderately until thereaction is complete or nearly complete. Insoluble compositions formedcan be obtained by filtering, while soluble compositions can be obtainedby evaporating the solvent. If acid groups on the chelating moietiesremain in the disclosed compositions, inorganic bases (e.g., hydroxides,carbonates and/or bicarbonates of sodium, potassium and/or lithium),organic bases, and/or basic amino acids can be used to neutralize acidicgroups, e.g., to facilitate isolation or purification of thecomposition.

In some embodiments, the detectable agent can be coupled to a TT1Peptide in such a way so as not to interfere with the ability of the TT1Peptide to interact with gC1qR/p32. In some embodiments, the detectableagent can be chemically bound to the TT1 Peptide. In some embodiments,the detectable agent can be chemically bound to a moiety that is itselfchemically bound to the De Roos et al., 1991, indirectly linking theimaging and targeting moieties.

II.F. Internalization Elements and Tissue Penetration Elements

The disclosed compositions, surface molecules, cargo molecules,peptides, proteins, amino acid sequences, etc. can comprise one or moreinternalization elements, tissue penetration elements, or both.Internalization elements and tissue penetration elements can beincorporated into or fused with other peptide components of thecomposition, such as peptide homing molecules and peptide cargomolecules. Internalization elements are molecules, often peptides oramino acid sequences, that allow the internalization element andcomponents with which it is associated, to pass through biologicalmembranes. Tissue penetration elements are molecules, often peptides oramino acid sequences, that allow the tissue penetration element andcomponents with which it is associated to passage into and throughtissue. “Internalization” refers to passage through a plasma membrane orother biological barrier. “Penetration” refers to passage into andthrough a membrane, cell, tissue, or other biological barrier.Penetration generally involves and includes internalization. Somemolecules, such as CendR elements, function as both internalizationelements and tissue penetration elements.

Internalization elements include, for example, cell-penetrating peptides(CPPs) and CendR peptides. Peptides that are internalized into cells arecommonly referred to as cell-penetrating peptides. There are two mainclasses of such peptides: hydrophobic and cationic (Zorko and Langel,2005). The cationic peptides, which are commonly used to introducenucleic acids, proteins into cells, include the prototypiccell-penetrating peptides (CPP), Tat, and penetratin (Derossi et al.,1998; Meade and Dowdy, 2007). A herpes virus protein, VP22, is capableof both entering and exiting cells and carrying a payload with it(Elliott and O'Hare, 1997; Brewis et al., 2003).

II.F.1. CendR Elements

Useful forms of internalization elements and tissue penetration elementsare CendR elements. CendR elements are amino acid sequences with aC-terminal element as a defining feature that signals highly efficientinternalization of phage and free peptides into cells. Thisinternalization phenomenon has been named the “C-end rule” or “CendR”.The CendR pathway can also be used for passage of compositions ofinterest from the vasculature and their spread into tissue. TheC-terminal element can cause spread of compositions from the vasculature(and thus can be spread into tumor tissue from an intravenous injection,for example). CendR elements can also be used to mediate passage ofcompositions of interest through other CendR-capable membranes, such asmucous membranes and the blood-brain barrier. As used herein, “tissuepenetration” and “penetration of tissue” refer to passage into orthrough a tissue beyond or through the outer or a first layer of cellsor through a tissue membrane. Such passage or penetration through tissue(which can also be referred to as extravasation and tissue penetration)can be a function of, for example, cell internalization and passagebetween cells in the tissue. Throughout this application, when the term“tissue penetration” is used, it is understood that such penetration canalso extend to other barriers and CendR-capable membranes foundthroughout the body, such as the blood brain barrier.

Unlike the known cell-penetrating peptides, the CendR internalizingelement is position-dependent-it is inactive when present in positionsother than the C-terminus of the peptide. Another distinguishing featureis that the CendR element is stereo-specific, that is, CendR elementscomposed of D-amino acids are inactive. A latent CendR peptide can beactivated by cleavage by, for example, the appropriate proteolyticenzyme to expose, for example, a C-terminal arginine, lysine, orlysine-glycine. Throughout the application, when the term “CendRelement” or “C-terminal element” is used, it is used to describe aC-terminal arginine, a C-terminal lysine, or a C-terminal lysine-glycinepair, where glycine is at the furthest C-terminal position. In otherwords, in the case where a lysine is on the C terminus end, the CendRelement can remain functional with a glycine on the C terminus side ofthe lysine. However, it is not necessary to have glycine on the end inorder for the lysine residue to be functional as a C-terminal element,so that lysine can be present without glycine and still be functional.The converse is not true, however, in that glycine cannot function as aC-terminal element without the presence of lysine adjacent to it.Arginine does not require either lysine or glycine to function as aC-terminal element, as long as it remains in the furthest C-terminalposition. Such CendR elements can be referred to as type 1 CendRelements.

The term “CendR element” or “C-terminal element” can also be used todescribe a C-terminal histidine and amino acid sequences having thesequence X₁X₂X₃X₄, where X₁ can be R, K or H, where X₄ can be R, K, H,or KG, and where X₂ and X₃ can each be, independently, any amino acid.Such CendR elements can be referred to as type 2 CendR elements. The X₂and X₃ amino acids can be selected for specific purposes. For example,X₂, X₃, or both can be chosen to form all or a portion of a proteaserecognition sequence. This would be useful, for example, to specify orenable cleavage of a peptide having the CendR element as a latent orcryptic CendR element that is activated by cleavage following the X₄amino acid. Examples of such amino acid choices are shown in Tables 3and 4. The X₁, X₂, and X₃ amino acids can also be selected, for example,to recruit additional proteins to NRP-1 molecules at the cell surface.This can be applied, for example, to modulate the selectivity andinternalization and/or tissue penetration potency of CendR elements (andthe compositions, conjugates, proteins, and peptides containing CendRelements). The X₂ and X₃ amino acids can also be selected to preventprotease cleavage within the X₁-X₄ motif. Optionally, certain aminoacids can also be excluded from use for X₂, X₃, or both. For example, ifdesired, G and D can be excluded from simultaneous use as X₂ and X₃,respectively. Some type 2 CendR elements can also be described asR/K/HXXR/K/H and R/K/HXXKG.

Examples of CendR elements include XXR/K/H, XXR/K, XXR/H, XXK/H, XXR,XXK, XXH, XXKG, RXXR/K/H, RXXR/K, RXXR/H, RXXK/H, RXXR, RXXK, RXH,RXXKG, KXXR/K/H, KXXR/K, KXXR/H, KXXK/H, KXXR, KXXK, KXXH, KXXKG,HXXR/K/H, HXXR/K, HXXR/H, HXXK/H, HXXR, HXXK, HXXH, HXXKG, R/K/HXXR,R/KXXR, R/HXXR, K/HXXR, RXXR, KXXR, HXXR, R/K/HXXK, R/KXXK, R/HXXK,K/HXXK, RXXK, KXXK, HXXK, R/K/HXXH, R/KXXH, R/HXXH, K/HXXH, RXXH, KXXH,HXXH, R/K/HXXKG, R/KXXKG, R/HXXKG, K/HXXKG, RXXKG, KXXKG, and HXXKG. ACendR element that can be internalized into a cell can be referred to asan internalization CendR element. A CendR element that can penetratetissue can be referred to as a penetrating CendR element. A CendRelement that can be internalized into a cell and that can penetratetissue can be referred to as an internalization and penetrating CendRelement. Unless the context clearly indicates otherwise, reference to“CendR element” refers to any of these, either individually,collectively, or in any combination.

As used herein, “CendR composition” refers to a composition thatcomprises a CendR element. The CendR element can be, for example,active, activatable, or blocked. For example, the CendR composition cancomprise a protein or peptide comprising an amino acid sequence thatcomprises a CendR element where the amino acid sequence is at theC-terminal end of the protein or peptide.

As used herein, “activatable CendR element” refers to a CendR elementhaving a molecule, moiety, nanoparticle, compound or other compositioncovalently coupled to the CendR element, such as to the terminalcarboxyl group of the C-terminal element, where the molecule, moiety,nanoparticle, compound or other composition can block internalizationand/or tissue penetration of the CendR composition, conjugate, molecule,protein, peptide, etc. and where the molecule, moiety, nanoparticle,compound or other composition can be removed (to expose the terminalcarboxy group, for example). For example, the activatable CendR elementcan be on the C-terminal end of the peptide, and can prevent the CendRelement from being internalized and/or from penetrating tissue. Themolecule, nanoparticle, moiety, compound or other composition covalentlycoupled to the CendR element can be referred to as the “blocking group.”For example, the blocking group can be coupled to the terminal carboxylgroup of the C-terminal arginine or lysine or other C-terminal aminoacid of the CendR element, to the C-terminal amino acid of the CendRelement, or to an amino acid of the CendR element other than theC-terminal amino acid. The blocking group can also be coupled, orassociated with a part of a CendR composition, conjugate, molecule,protein, peptide, etc. other than the CendR element so long as it canprevent the CendR element from being internalized and/or frompenetrating tissue. A CendR composition comprising an activatable CendRelement can be referred to as an activatable CendR composition. A CendRmolecule comprising an activatable CendR element can be referred to asan activatable CendR molecule. A CendR conjugate comprising anactivatable CendR element can be referred to as an activatable CendRconjugate. A CendR protein comprising an activatable CendR element canbe referred to as an activatable CendR protein. A CendR peptidecomprising an activatable CendR element can be referred to as anactivatable CendR peptide.

An activatable CendR element can be blocked from internalization into acell, from tissue penetration, or both. Generally, an activatable CendRelement will be blocked from both internalization into a cell andpenetration of tissue. Such activatable CendR elements can be referredto as activatable internalization and penetrating CendR elements.However, some activatable CendR elements could be blocked only fromtissue penetration or only from internalization into a cell. Suchactivatable CendR elements can be referred to as activatableinternalization CendR elements (for CendR elements that are blocked onlyfrom internalization into a cell) or as activatable internalization andpenetrating CendR elements (for CendR elements that are blocked onlyfrom penetration of tissue). Generally, internalization CendR elementsthat are activatable will be activatable internalization CendR elements.Similarly, penetrating CendR elements that are activatable generallywill be activatable penetrating CendR elements. Internalization andpenetrating CendR elements that are activatable will be activatableinternalization and penetrating CendR elements. Removal of the blockinggroup will allow the CendR element to be internalized into a cell,penetrate tissue, or both.

The cleavable bond of an activatable CendR element can be cleaved in anysuitable way. For example, the cleavable bond can be cleavedenzymatically or non-enzymatically. For enzymatic cleavage, the cleavingenzyme can be supplied or can be present at a site where the CendRelement is delivered, homes, travels or accumulates. For example, theenzyme can be present in proximity to a cell to which the CendR elementis delivered, homes, travels, or accumulates. For non-enzymaticcleavage, the CendR element can be brought into contact with a cleavingagent, can be placed in cleaving conditions, or both. A cleaving agentis any substance that can mediate or stimulate cleavage of the cleavablebond. A non-enzymatic cleaving agent is any cleaving agent exceptenzymes. Cleaving conditions can be any solution or environmentalconditions that can mediate or stimulate cleavage of the cleavable bond.For example, some labile bonds can be cleaved in acid conditions,alkaline conditions, in the presence of a reactive group, etc.Non-enzymatic cleaving conditions are any cleaving conditions except thepresence of enzymes. Non-agent cleaving conditions are any cleavingconditions except the presence of cleaving agents.

A “protease-activatable CendR element” (or “protease-activated CendRelement”) refers to an activatable CendR element where the blockinggroup is coupled to the CendR element via a peptide bond and where thepeptide bond can be cleaved by a protease. Cleavage of this peptide bondin a protease-activatable CendR element makes the CendR element capableof internalization into a cell and/or of tissue penetration. In oneexample, the blocking group can be coupled to the CendR element via acleavable or labile bond. The cleavable bond can be cleaved by, forexample, an enzyme or a chemical compound. Cleavage or ‘labilization’bond in an activatable CendR element makes the CendR element capable ofinternalization into a cell and/or of tissue penetration. Such cleavageor ‘labilization’ can be referred to as activation of the CendR element.A protease-activatable CendR element is a form of activatable CendRelement.

Proteolysis that uncovers a C-terminal element can serve as a switchthat triggers the internalization signal. Various compositions can beinternalized through this mechanism. For example, homingmolecule-mediated accumulation can occur at a target site with celltype-specific proteolysis that exposes a C-terminal element which allowsfor highly specific homing systems with target-triggeredinternalization. This protease-controllable internalization system canbe useful in engineering compositions with functions such as celltype-specific and/or tissue type-specific uptake and the ability tospread the compositions in tissues.

CendR elements are further described in U.S. Patent ApplicationPublication Nos. 2009/0226372 and 2010/0322862, which are herebyincorporated by reference in their entirety, and specifically for theirdescription of the form, structure, and use of CendR elements andpeptides.

II.G. Surface Molecules

The surface molecules, alternatively referred to as a surface particles,disclosed herein can be conjugated with homing molecules and cargomolecules in such a way that the composition is delivered to a target.The surface molecule can be any substance that can be used with thehoming molecules and cargo molecules, and is not restricted by size orsubstance. Examples include, but are not limited to, nanoparticles (suchas iron oxide nanoparticles or albumin nanoparticles), liposomes, smallorganic molecules, microparticles, or microbubbles, such as fluorocarbonmicrobubbles. The term surface molecule is used to identify a componentof the disclosed composition but is not intended to be limiting. Inparticular, the disclosed surface molecules are not limited tosubstances, compounds, compositions, particles or other materialscomposed of a single molecule. Rather, the disclosed surface moleculesare any substance(s), compound(s), composition(s), particle(s) and/orother material(s) that can be conjugated with a plurality of homingmolecules and cargo molecules such that at least some of the homingmolecules and/or cargo molecules are presented and/or accessible on thesurface of the surface molecule. A variety of examples of suitablesurface molecules are described and disclosed herein.

The surface molecule can be detectable, or can be a therapeutic agentsuch as iRGD, RGD, or Abraxane™. The section herein which discussescargo molecules and moieties that can be detectable or therapeutic alsoapplies to the surface molecule.

Surface molecules can be associated with and arranged in thecompositions in a variety of configurations. In some embodiments,surface molecules can be associated with, conjugated to, and/orcovalently coupled to a plurality of homing molecules, a plurality ofcargo molecules, or both. In some embodiments, surface molecules can beassociated with, conjugated to, and/or covalently coupled to a pluralityof homing molecules, wherein the homing molecules can be associatedwith, conjugated to, and/or covalently coupled to a plurality of cargomolecules. In some embodiments, surface molecules can be associatedwith, conjugated to, and/or covalently coupled to a plurality of cargomolecules, wherein the cargo molecules can be associated with,conjugated to, and/or covalently coupled to a plurality of homingmolecules. Combinations of these combinations can also be used.

II.G.1. Nanoparticles, Microparticles, and Microbubbles

The term “nanoparticle” refers to a nanoscale particle with a size thatis measured in nanometers, for example, a nanoscopic particle that hasat least one dimension of less than about 100 nm. Examples ofnanoparticles include paramagnetic nanoparticles, superparamagneticnanoparticles, metal nanoparticles, nanoworms, fullerene-like materials,inorganic nanotubes, dendrimers (such as with covalently attached metalchelates), nanofibers, nanohoms, nano-onions, nanorods, nanoropes andquantum dots. A nanoparticle can produce a detectable signal, forexample, through absorption and/or emission of photons (including radiofrequency and visible photons) and plasmon resonance.

Microspheres (or microbubbles) can also be used with the methodsdisclosed herein. Microspheres containing chromophores have beenutilized in an extensive variety of applications, including photoniccrystals, biological labeling, and flow visualization in microfluidicchannels. See e.g., Lin et al., 2002; Wang et al., 2003; Gao et al.,2002; Han et al., 2001; Pai et al., 1999, each of which is incorporatedby reference in its entirety. Both the photostability of thechromophores and the monodispersity of the microspheres can beimportant.

Nanoparticles, such as, for example, metal nanoparticles, metal oxidenanoparticles, or semiconductor nanocrystals can be incorporated intomicrospheres. The optical, magnetic, and electronic properties of thenanoparticles can allow them to be observed while associated with themicrospheres and can allow the microspheres to be identified andspatially monitored. For example, the high photostability, goodfluorescence efficiency and wide emission tunability of colloidallysynthesized semiconductor nanocrystals can make them an excellent choiceof chromophore. Unlike organic dyes, nanocrystals that emit differentcolors (i.e. different wavelengths) can be excited simultaneously with asingle light source. Colloidally synthesized semiconductor nanocrystals(such as, for example, core-shell CdSe/ZnS and CdS/ZnS nanocrystals) canbe incorporated into microspheres. The microspheres can be monodispersesilica microspheres.

The nanoparticle can be a metal nanoparticle, a metal oxidenanoparticle, or a semiconductor nanocrystal. The metal of the metalnanoparticle or the metal oxide nanoparticle can include titanium,zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum,tungsten, manganese, technetium, rhenium, iron, ruthenium, osmium,cobalt, rhodium, iridium, nickel, palladium, platinum, copper, silver,gold, zinc, cadmium, scandium, yttrium, lanthanum, a lanthanide seriesor actinide series element (e.g., cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium,and uranium), boron, aluminum, gallium, indium, thallium, silicon,germanium, tin, lead, antimony, bismuth, polonium, magnesium, calcium,strontium, and barium. In certain embodiments, the metal can be iron,ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver, gold,cerium or samarium. The metal oxide can be an oxide of any of thesematerials or combination of materials. For example, the metal can begold, or the metal oxide can be an iron oxide, a cobalt oxide, a zincoxide, a cerium oxide, or a titanium oxide. Preparation of metal andmetal oxide nanoparticles is described, for example, in U.S. Pat. Nos.5,897,945 and 6,759,199, each of which is incorporated by reference inits entirety.

The nanoparticles can be comprised of cargo molecules and a carrierprotein (such as albumin). Such nanoparticles are useful, for example,to deliver hydrophobic or poorly soluble compounds. Nanoparticles ofpoorly water soluble drugs (such as taxane) have been disclosed in, forexample, U.S. Pat. Nos. 5,916,596; 6,506,405; and 6,537,579; and alsoU.S. Patent Application Publication No. 2005/0004002.

In forms, the nanoparticles can have an average or mean diameter of nogreater than about 1000 nanometers (nm), such as no greater than aboutany of 900, 800, 700, 600, 500, 400, 300, 200, and 100 nm. In someembodiments, the average or mean diameters of the nanoparticles can beno greater than about 200 nm. In some embodiments, the average or meandiameters of the nanoparticles can be no greater than about 150 nm. Insome embodiments, the average or mean diameters of the nanoparticles canbe no greater than about 100 nm. In some embodiments, the average ormean diameter of the nanoparticles can be about 20 to about 400 nm. Insome embodiments, the average or mean diameter of the nanoparticles canbe about 40 to about 200 nm. In some embodiments, the nanoparticles aresterile-filterable.

The nanoparticles can be present in a dry formulation (such aslyophilized composition) or suspended in a biocompatible medium.Suitable biocompatible media include, but are not limited to, water,buffered aqueous media, saline, buffered saline, optionally bufferedsolutions of amino acids, optionally buffered solutions of proteins,optionally buffered solutions of sugars, optionally buffered solutionsof vitamins, optionally buffered solutions of synthetic polymers,lipid-containing emulsions, and the like.

Examples of suitable carrier proteins include proteins normally found inblood or plasma, which include, but are not limited to, albumin,immunoglobulin including IgA, lipoproteins, apolipoprotein B, alpha-acidglycoprotein, beta-2-macroglobulin, thyroglobulin, transferin,fibronectin, factor VII, factor VIII, factor IX, factor X, and the like.In some embodiments, the carrier protein is non-blood protein, such ascasein, α-lactalbumin, and β-lactoglobulin. The carrier proteins mayeither be natural in origin or synthetically prepared. In someembodiments, the pharmaceutically acceptable carrier comprises albumin,such as human serum albumin. Human serum albumin (HSA) is a highlysoluble globular protein of Mr 65K and consists of 585 amino acids. HSAis the most abundant protein in the plasma and accounts for 70-80% ofthe colloid osmotic pressure of human plasma. The amino acid sequence ofHSA contains a total of 17 disulfide bridges, one free thiol (Cys 34),and a single tryptophan (Trp 214). Intravenous use of HSA solution hasbeen indicated for the prevention and treatment of hypovolumic shock(see e.g., Tullis, 1977; Houser et al., 1980) and in conjunction withexchange transfusion in the treatment of neonatal hyperbilirubinemia(see e.g., Finlayson, 1980). Other albumins are contemplated, such asbovine serum albumin. Use of such non-human albumins could beappropriate, for example, in the context of use of these compositions innon-human mammals, such as the veterinary (including domestic pets andagricultural context).

Carrier proteins (such as albumin) in the composition generally serve asa carrier for the hydrophobic cargo molecules, i.e., the carrier proteinin the composition makes the cargo molecules more readily suspendable inan aqueous medium or helps maintain the suspension as compared tocompositions not comprising a carrier protein. This can avoid the use oftoxic solvents (or surfactants) for solubilizing the cargo molecules,and thereby can reduce one or more side effects of administration of thecargo molecules into an individual (such as a human). Thus, in someembodiments, the composition described herein can be substantially free(such as free) of surfactants, such as Cremophor (including CREMOPHOREL® (BASF)). In some embodiments, the composition can be substantiallyfree (such as free) of surfactants. A composition is “substantially freeof Cremophor” or “substantially free of surfactant” if the amount ofCremophor or surfactant in the composition is not sufficient to causeone or more side effect(s) in an individual when the composition isadministered to the individual.

The amount of carrier protein in the composition described herein willvary depending on other components in the composition. In someembodiments, the composition comprises a carrier protein in an amountthat is sufficient to stabilize the cargo molecules in an aqueoussuspension, for example, in the form of a stable colloidal suspension(such as a stable suspension of nanoparticles). In some embodiments, thecarrier protein is in an amount that reduces the sedimentation rate ofthe cargo molecules in an aqueous medium. For particle-containingcompositions, the amount of the carrier protein also depends on the sizeand density of nanoparticles of the cargo molecules.

Methods of making nanoparticle compositions are known in the art. Forexample, nanoparticles containing cargo molecules and carrier protein(such as albumin) can be prepared under conditions of high shear forces(e.g., sonication, high pressure homogenization, or the like). Thesemethods are disclosed in, for example, U.S. Pat. Nos. 5,916,596;6,506,405; 6,537,579; see also U.S. Patent Application Publication No.2005/0004002.

Briefly, the hydrophobic carrier molecules can be dissolved in anorganic solvent, and the solution can be added to a human serum albuminsolution. The mixture is subjected to high pressure homogenization. Theorganic solvent can then be removed by evaporation. The dispersionobtained can be further lyophilized. Suitable organic solvent include,for example, ketones, esters, ethers, chlorinated solvents, and othersolvents known in the art. For example, the organic solvent can bemethylene chloride and chloroform/ethanol (for example with a ratio of1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, or 9:1).

The nanoparticle can also be, for example, a heat generating nanoshell.As used herein, “nanoshell” is a nanoparticle having a discretedielectric or semi-conducting core section surrounded by one or moreconducting shell layers. U.S. Pat. No. 6,530,944 is hereby incorporatedby reference herein in its entirety for its teaching of the methods ofmaking and using metal nanoshells. Targeting molecules can be attachedto the disclosed compositions and/or carriers. For example, thetargeting molecules can be antibodies or fragments thereof, ligands forspecific receptors, or other proteins specifically binding to thesurface of the cells to be targeted.

II.G.2. Liposomes

“Liposome” as the term is used herein refers to a structure comprisingan outer lipid bi- or multi-layer membrane surrounding an internalaqueous space. Liposomes can be used to package any biologically activeagent for delivery to cells.

Materials and procedures for forming liposomes are well-known to thoseskilled in the art. Upon dispersion in an appropriate medium, a widevariety of phospholipids swell, hydrate and form multilamellarconcentric bilayer vesicles with layers of aqueous media separating thelipid bilayers. These systems are referred to as multilamellar liposomesor multilamellar lipid vesicles (“MLVs”) and have diameters within therange of 10 nm to 100 am. These MLVs were first described by Bangham etal., 1965. In general, lipids or lipophilic substances are dissolved inan organic solvent. When the solvent is removed, such as under vacuum byrotary evaporation, the lipid residue forms a film on the wall of thecontainer. An aqueous solution that typically contains electrolytes orhydrophilic biologically active materials is then added to the film.Large MLVs are produced upon agitation. When smaller MLVs are desired,the larger vesicles are subjected to sonication, sequential filtrationthrough filters with decreasing pore size or reduced by other forms ofmechanical shearing. There are also techniques by which MLVs can bereduced both in size and in number of lamellae, for example, bypressurized extrusion (Barenholz et al., 1979).

Liposomes can also take the form of unilamnellar vesicles, which areprepared by more extensive sonication of MLVs, and consist of a singlespherical lipid bilayer surrounding an aqueous solution. Unilamellarvesicles (“ULVs”) can be small, having diameters within the range of 20to 200 nm, while larger ULVs can have diameters within the range of 200nm to 2 am. There are several well-known techniques for makingunilamellar vesicles. In Papahadjopoulos et al., 1968, sonication of anaqueous dispersion of phospholipids produces small ULVs having a lipidbilayer surrounding an aqueous solution. Schneider, U.S. Pat. No.4,089,801 describes the formation of liposome precursors byultrasonication, followed by the addition of an aqueous mediumcontaining amphiphilic compounds and centrifugation to form abiomolecular lipid layer system.

Small ULVs can also be prepared by the ethanol injection techniquedescribed by Batzri et al., 1973 and the ether injection technique ofDeamer et al., 1976. These methods involve the rapid injection of anorganic solution of lipids into a buffer solution, which results in therapid formation of unilamellar liposomes. Another technique for makingULVs is taught by Weder et al., 1984. This detergent removal methodinvolves solubilizing the lipids and additives with detergents byagitation or sonication to produce the desired vesicles.

U.S. Pat. No. 4,235,871 describes the preparation of large ULVs by areverse phase evaporation technique that involves the formation of awater-in-oil emulsion of lipids in an organic solvent and the drug to beencapsulated in an aqueous buffer solution. The organic solvent isremoved under pressure to yield a mixture which, upon agitation ordispersion in an aqueous media, is converted to large ULVs. U.S. Pat.No. 4,016,100 describes another method of encapsulating agents inunilamellar vesicles by freezing/thawing an aqueous phospholipiddispersion of the agent and lipids.

In addition to the MLVs and ULVs, liposomes can also be multivesicular.Described in Kim et al., 1983, these multivesicular liposomes arespherical and contain internal granular structures. The outer membraneis a lipid bilayer and the internal region contains small compartmentsseparated by bilayer septum. Still yet another type of liposomes areoligolamellar vesicles (“OLVs”), which have a large center compartmentsurrounded by several peripheral lipid layers. These vesicles, having adiameter of 2-15 jam, are described in Callo et al., 1985.

U.S. Pat. Nos. 4,485,054 and 4,761,288 also describe methods ofpreparing lipid vesicles. More recently, Hsu, U.S. Pat. No. 5,653,996describes A method for preparing liposomes utilizing aerosolization andU.S. Pat. No. 5,013,497 describes a method for preparing liposomesutilizing a high velocity-shear mixing chamber. Methods are alsodescribed that use specific starting materials to produce ULVs (U.S.Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848 and5,628,936).

A comprehensive review of all the aforementioned lipid vesicles andmethods for their preparation are described in Gregoriadis, 1984. Thisand the aforementioned references describing various lipid vesiclessuitable for use in the invention are incorporated herein by reference.

II.G.3. Micelles

“Micelle” as used herein refers to a structure comprising an outer lipidmonolayer. Micelles can be formed in an aqueous medium when the CriticalMicelle Concentration (CMC) is exceeded. Small micelles in dilutesolution at approximately the critical micelle concentration (CMC) aregenerally believed to be spherical. However, under other conditions,they may be in the shape of distorted spheres, disks, rods, lamellae,and the like.

Micelles formed from relatively low molecular weight amphiphilemolecules can have a high CMC so that the formed micelles dissociaterather rapidly upon dilution. If this is undesired, amphiphile moleculeswith large hydrophobic regions can be used. For example, lipids with along fatty acid chain or two fatty acid chains, such as phospholipidsand sphingolipids, or polymers, specifically block copolymers, can beused.

Polymeric micelles have been prepared that exhibit CMCs as low as 10⁻⁶ M(molar). Thus, they tend to be very stable while at the same timeshowing the same beneficial characteristics as amphiphile micelles. Anymicelle-forming polymer presently known in the art or as such may becomeknown in the future may be used in the disclosed compositions andmethods. Examples of micelle-forming polymers include, withoutlimitation, methoxy poly(ethylene glycol)-b-poly(ε-caprolactone),conjugates of poly(ethylene glycol) with phosphatidyl-ethanolamine,poly(ethylene glycol)-b-polyesters, poly(ethyleneglycol)-b-poly(L-aminoacids),poly(N-vinylpyrrolidone)-bl-poly(orthoesters),poly(N-vinylpyrrolidone)-b-polyanhydrides andpoly(N-vinylpyrrolidone)-b-poly(alkyl acrylates).

Micelles can be produced by processes conventional in the art. Examplesof such are described in, for example, Liggins & Burt, 2002; Zhang etal., 1996; U.S. Pat. No. 4,745,160. In one such method,polyether-polyester block copolymers, which are amphipathic polymershaving hydrophilic (polyether) and hydrophobic (polyester) segments, areused as micelle forming carriers.

Another type of micelle can be formed using, for example, AB-type blockcopolymers having both hydrophilic and hydrophobic segments, asdescribed in, for example, Tuzar & Kratochvil, 1976; Wilhelm et al.,1991. These polymeric micelles are able to maintain satisfactory aqueousstability. These micelles, in the range of approximately <200 nm insize, are effective in reducing non-selective RES scavenging and showenhanced permeability and retention.

Further, U.S. Pat. No. 5,929,177 to Kataoka et al. describes a polymericmolecule which is usable as, inter alia, a drug delivery carrier. Themicelle is formed from a block copolymer having functional groups onboth of its ends and which comprises hydrophilic/hydrophobic segments.The polymer functional groups on the ends of the block copolymer includeamino, carboxyl and mercapto groups on the .alpha.-terminal andhydroxyl, carboxyl group, aldehyde group and vinyl group on the.omega.-terminal. The hydrophilic segment comprises polyethylene oxide,while the hydrophobic segment is derived from lactide, lactone or(meth)acrylic acid ester.

Further, for example, poly(D,L-lactide)-b-methoxypolyethylene glycol(MePEG:PDLLA) diblock copolymers can be made using MePEG 1900 and 5000.The reaction can be allowed to proceed for 3 hr at 160° C., usingstannous octoate (0.25%) as a catalyst. However, a temperature as low as130° C. can be used if the reaction is allowed to proceed for about 6hr, or a temperature as high as 190° C. can be used if the reaction iscarried out for only about 2 hr.

As another example, N-isopropylacrylamide (IPAAm; Kohjin, Tokyo, Japan)and dimethylacrylamide (DMAAm; Wako Pure Chemicals, Tokyo, Japan) can beused to make hydroxyl-terminated poly(IPAAm-co-DMAAm) in a radicalpolymerization process, using the method of Kohori et al., 1998. Theobtained copolymer can be dissolved in cold water and filtered throughtwo ultrafiltration membranes with a 10,000 and 20,000 molecular weightcut-off. The polymer solution is first filtered through a 20,000molecular weight cut-off membrane. Then the filtrate was filtered againthrough a 10,000 molecular weight cut-off membrane. Three molecularweight fractions can be obtained as a result, a low molecular weight, amiddle molecular weight, and a high molecular weight fraction. A blockcopolymer can then be synthesized by a ring opening polymerization ofD,L-lactide from the terminal hydroxyl group of the poly(IPAAm-co-DMAAm)of the middle molecular weight fraction. The resultingpoly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) copolymer can be purified asdescribed in Kohori et al., 1999.

Examples of block copolymers from which micelles can be prepared whichcan be used to coat a support surface are found in U.S. Pat. No.5,925,720 to Kataoka et al.; U.S. Pat. No. 5,412,072 to Sakarai et al.;U.S. Pat. No. 5,410,016 to Kataoka et al.; U.S. Pat. No. 5,929,177 toKataoka et al.; U.S. Pat. No. 5,693,751 to Sakurai et al.; U.S. Pat. No.5,449,513 to Yokoyama et al.; PCT International Patent ApplicationPublication Nos. WO 96/32434; WO 96/33233; WO 97/0623, the contents ofall of which are incorporated by reference. Modifications thereof whichare prepared by introducing thereon a suitable functional group(including an ethyleneically unsaturated polymerizable group) are alsoexamples of block copolymers from which micelles of the presentinvention are preferably prepared. Preferable block copolymers are thosedisclosed in the above-mentioned patents and or international patentpublications. If the block copolymer has a sugar residue on one end ofthe hydrophilic polymer segment, as in the block copolymer of WO96/32434, the sugar residue should preferably be subjected to Malapradeoxidation so that a corresponding aldehyde group may be formed.

II.G.4. Lipids

Lipids are synthetically or naturally-occurring molecules which includesfats, waxes, sterols, prenol lipids, fat-soluble vitamins (such asvitamins A, D, E and K), glycerolipids, monoglycerides, diglycerides,triglycerides, glycerophospholipids, sphingolipids, phospholipids, fattyacids monoglycerides, saccharolipids and others. Lipids can behydrophobic or amphiphilic small molecules; the amphiphilic nature ofsome lipids allows them to form structures such as monolayers, vesicles,micelles, liposomes, bi-layers or membranes in an appropriateenvironment i.e. aqueous environment. Any of a number of lipids can beused as amphiphile molecules, including amphipathic, neutral, cationic,and anionic lipids. Such lipids can be used alone or in combination, andcan also include bilayer stabilizing components such as polyamideoligomers (see e.g., U.S. Pat. No. 6,320,017), peptides, proteins,detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see e.g., U.S.Pat. No. 5,885,613). In some embodiments, cloaking agents, which reduceelimination of liposomes by the host immune system, can also beincluded, such as polyamide-oligomer conjugates, e.g., ATTA-lipids, (seee.g., U.S. patent application Ser. No. 08/996,783, filed Feb. 2, 1998)and PEG-lipid conjugates (see e.g., U.S. Pat. Nos. 5,820,873; 5,534,499;and 5,885,613).

Any of a number of neutral lipids can be included, referring to any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH, includingdiacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

Cationic lipids, carry a net positive charge at physiological pH, canreadily be used as amphiphile molecules. Such lipids include, but arenot limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy) propyl-N,N—N-triethylammonium chloride (“DOTMA”);N,N-di stearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);3.beta.-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol(“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as LIPOFECTIN (including DOTMA andDOPE, available from GIBCO/BRL), LIPOFECTAMINE (comprising DOSPA andDOPE, available from GIBCO/BRL), and TRANSFECTAM (comprising DOGS, inethanol, from Promega Corp.).

Anionic lipids can be used as amphiphile molecules and include, but arenot limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanol amine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

Amphiphatic lipids can also be suitable amphiphile molecules.“Amphipathic lipids” refer to any suitable material, wherein thehydrophobic portion of the lipid material orients into a hydrophobicphase, while the hydrophilic portion orients toward the aqueous phase.Such compounds include, but are not limited to, fatty acids,phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatdylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and 3-acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols. Zwitterionic lipids are aform of amphiphatic lipid.

Sphingolipids are fatty acids conjugated to the aliphatic amino alcoholsphingosine. The fatty acid can be covalently bond to sphingosine via anamide bond. Any amino acid as described above can be covalently bond tosphingosine to form a sphingolipid. A sphingolipid can be furthermodified by covalent bonding through the α-hydroxyl group. Themodification can include alkyl groups, alkenyl groups, alkynyl groups,aromatic groups, heteroaromatic groups, cyclyl groups, heterocyclylgroups, phosphonic acid groups. Non-limiting examples of shingolipidsare N-acylsphingosine, N-Acylsphingomyelin, Forssman antigen.

Saccharolipids are compounds that contain both fatty acids and sugars.The fatty acids are covalently bonded to a sugar backbone. The sugarbackbone can contain one or more sugars. The fatty acids can bond to thesugars via either amide or ester bonds. The sugar can be any sugar base.The fatty acid can be any fatty acid as described elsewhere herein. Theprovided compositions can comprise either natural or syntheticsaccharolipids. Non-limiting saccharolipids areUDP-3-O-(β-hydroxymyristoyl)-GlcNAc, lipid IV A, Kdo2-lipid A.

II.H. Linkers

Disclosed are linkers for associating components of the disclosedcompositions. Such linkers can be any molecule, conjugate, composition,etc. that can be used to associate components of the disclosedcompositions. Generally, linkers can be used to associate componentsother than surface molecules to surface molecules. Useful linkersinclude materials that are biocompatible, have low bioactivity, have lowantigenicity, etc. That is, such useful linker materials can serve thelinking/association function without adding unwanted bioreactivity tothe disclosed compositions. Many such materials are known and used forsimilar linking and association functions. Polymer materials are aparticularly useful form of linker material. For example, polyethyleneglycols can be used.

Linkers are useful for achieving useful numbers and densities of thecomponents (such as homing molecules and membrane perturbing molecules)on surface molecules. For example, linkers of fibrous form are usefulfor increasing the number of components per surface molecule or per agiven area of the surface molecule. Similarly, linkers having abranching form are useful for increasing the number of components persurface molecule or per a given area of the surface molecule. Linkerscan also have a branching fibrous form.

Linkers of different lengths can be used to bind the disclosedcomponents to surface molecules and to each other. A flexible linker canfunction well even if relatively short, while a stiffer linker may canbe longer to allow effective exposure and density. The length of alinker can refer to the number of atoms in a continuous covalent chainbetween the attachment points on the components being linked or to thelength (in nanometers, for example) of a continuous covalent chainbetween the attachment points on the components being linked. Unless thecontext clearly indicates otherwise, the length refers to the shortestcontinuous covalent chain between the attachment points on thecomponents being linked not accounting for side chains, branches, orloops. Due to flexibility of the linker, all of the linkers may not havesame distance from the surface molecule. Thus linkers with differentchain lengths can make the resulting composition more effective (byincreasing density, for example). Branched linkers bearing multiplecomponents also allow attachment of more than one component at a givensite of the surface molecule. Useful lengths for linkers include atleast, up to, about, exactly, or between 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000atoms. Useful lengths for linkers include at least, up to, about,exactly, or between 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 150, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1,000, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nanometers. Any range ofthese lengths and all lengths between the listed lengths arespecifically contemplated.

Hydrophilic or water-solubility linkers can increase the mobility of theattached components. Examples of water-soluble, biocompatible polymerswhich can serve as linkers include, but are not limited to polymers suchpolyethylene glycol (PEG), polyethylene oxide (PEO), polyvinyl alcohol,polyhydroxyethyl methacrylate, polyacrylamide, and natural polymers suchas hyaluronic acid, chondroitin sulfate, carboxymethylcellulose, andstarch. Useful forms of branched tethers include star PEO and comb PEO.Star PEO can be formed of many PEO “arms” emanating from a common core.

Polyethylene glycols (PEGs) are simple, neutral polyethers which havebeen given much attention in biotechnical and biomedical applications(Harris, 1992). PEGs are soluble in most solvents, including water, andare highly hydrated in aqueous environments, with two or three watermolecules bound to each ethylene glycol segment; this hydrationphenomenon has the effect of preventing adsorption either of otherpolymers or of proteins onto PEG-modified surfaces. Furthermore, PEGsmay readily be modified and bound to other molecules with only littleeffect on their chemistry. Their advantageous solubility and biologicalproperties are apparent from the many possible uses of PEGs andcopolymers thereof, including block copolymers such as PEG-polyurethanesand PEG-polypropylenes. Appropriate molecular weights for PEG linkersused in the disclosed compositions can be from about 120 daltons (Da) toabout 20 kilodaltons (kDa). For example, PEGs can be at least, up to,about, exactly, or between 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10,000, 20,000, 30,000, 40,000, and 50,000 daltons. Anyrange of these masses and all masses between the listed masses arespecifically contemplated. PEGs are usually available as mixtures ofsomewhat heterogeneous masses with a stated average mass (PEG-5000, forexample).

The disclosed compositions can be produced using any suitabletechniques. Many techniques, reactive groups, chemistries, etc. forlinking components of the types disclosed herein are known and can beused with the disclosed components and compositions. Examples of sometechniques for producing the disclosed compositions are described in theexamples.

Protein crosslinkers that can be used to crosslink other molecules,elements, moieties, etc. to the disclosed compositions, surfacemolecules, homing molecules, membrane perturbing molecules,internalization elements, tissue penetration elements, cargocompositions, CendR elements, compositions, proteins, peptides, aminoacid sequences, etc. are known in the art and are defined based onutility and structure and include DSS (Disuccinimidylsuberate), DSP(Dithiobis(succinimidylpropionate)), DTSSP (3,3′-Dithiobis(sulfosuccinimidylpropionate)), SULFO BSOCOES(Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS(Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethyleneglycolbis(sulfosuccinimidylsuccinate)), DPDPB(1,2-Di[3′-(2′-pyridyldithio) propionamido]butane), BSSS(Bis(sulfosuccinimdyl) suberate), SMPB(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB(N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC(Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SULFOSMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate),NHS LC SPDP (Succinimidyl-6-[3-(2-pyridyldithio) propionamido)hexanoate), SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio)propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHSIODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH(4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid hydrazidehydrochloride), MBH (m-Maleimidobenzoic acid hydrazidehydrochloride),SULFO EMCS (N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI(N-(p-Maleimidophenyl) isocyanate), KMUH (N-(kappa-Maleimidoundecanoicacid) hydrazide), LC SMCC(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate)),SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide ester), SMPH(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS(N-(kappa-maleimidoundecanoyloxy) sulfosuccinimide ester), GMBS(N-(gamma-Maleimidobutyrloxy) succinimide), DMP (Dimethylpimelimidatehydrochloride), DMS (Dimethyl suberimidate hydrochloride), MHBH (Wood'sReagent; Methyl-p-hydroxybenzimidate hydrochloride, 98%), DMA(Dimethyladipimidate hydrochloride).

Components of the disclosed compositions, such as surface molecules,homing molecules, membrane perturbing molecules, internalizationelements, tissue penetration elements, etc., can also be coupled using,for example, maleimide coupling. By way of illustration, components canbe coupled to lipids by coupling to, for example,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000; DSPE-PEG2000-maleimide] (Avanti Polar Lipids) by making useof a free cysteine sulfhydryl group on the component. The reaction canbe performed, for example, in aqueous solution at room temperature for 4hours. This coupling chemistry can be used to couple components ofco-compositions and cargo compositions.

Components of the disclosed compositions, such as surface molecules,homing molecules, membrane perturbing molecules, internalizationelements, tissue penetration elements, etc., can also be coupled using,for example, amino group-functionalized dextran chemistry. Particles,such as, for example, nanoparticles, nanoworms, and micelles, can becoated with amino group functionalized dextran. Attachment of PEG toaminated particles increases the circulation time, presumably byreducing the binding of plasma proteins involved in opsonization(Moghimi et al., 2001). The particles can have surface modifications,for example, for reticuloendothelial system avoidance (PEG) and homing(homing molecules), endosome escape (pH-sensitive peptide; for example,Pirollo et al., 2007), a detectable agent, a therapeutic compound, or acombination. To accommodate all these functions on one particle,optimization studies can be conducted to determine what proportion ofthe available linking sites at the surface of the particles any one ofthese elements should occupy to give the best combination of targetingand payload delivery. The cell internalization and/or tissue penetrationof such compositions can be mediated by the disclosed CendR elements,amino acid sequences, peptides, proteins, molecules, conjugates, andcompositions.

The provided peptides and polypeptides can have additional N-terminal,C-terminal, or intermediate amino acid sequences, e.g., amino acidlinkers or tags. The term “amino acid linker” refers to an amino acidsequences or insertions that can be used to connect or separate twodistinct peptides, polypeptides, or polypeptide fragments, where thelinker does not otherwise contribute to the essential function of thecomposition. The term “amino acid tag” refers to a distinct amino acidsequence that can be used to detect or purify the provided polypeptide,wherein the tag does not otherwise contribute to the essential functionof the composition. The provided peptides and polypeptides can furtherhave deleted N-terminal, C-terminal or intermediate amino acids that donot contribute to the essential activity of the peptides andpolypeptides.

Components can be directly or indirectly covalently bound to surfacemolecules or each other by any functional group (e.g., amine, carbonyl,carboxyl, aldehyde, alcohol). For example, one or more amine, alcohol orthiol groups on the components can be reacted directly withisothiocyanate, acyl azide, N-hydroxysuccinimide ester, aldehyde,epoxide, anhydride, lactone, or other functional groups incorporatedonto the surface molecules or other components. Schiff bases formedbetween the amine groups on the components and aldehyde groups on thesurface molecule or other components can be reduced with agents such assodium cyanoborohydride to form hydrolytically stable amine links(Ferreira et al., 2003). Components can be coupled to surface moleculesand other components by, for example, the use of a heterobifunctionalsilane linker reagent, or by other reactions that activate functionalgroups on either the surface molecule or the components.

Useful modes for linking components to surface molecules and to othercomponents include heterobifunctional linkers or spacers. Such linkerscan have both terminal amine and thiol reactive functional groups forreacting amines on components with sulfhydryl groups, thereby couplingthe components in an oriented way. These linkers can contain a variablenumber of atoms. Examples of such linkers include, but are not limitedto, N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP, 3- and 7-atomspacer), long-chain-SPDP (12-atom spacer),(Succinimidyloxycarbonyl-a-methyl-2-(2-pyridyldithio) toluene) (SMPT,8-atom spacer),Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC,11-atom spacer) andSulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,(sulfo-SMCC, 11-atom spacer), m-Maleimidobenzoyl-N hydroxysuccinimideester (MBS, 9-atom spacer), N-(g-maleimidobutyryloxy)succinimide ester(GMBS, 8-atom spacer), N-(g-maleimidobutyryloxy) sulfosuccinimide ester(sulfo-GMBS, 8-atom spacer), Succinimidyl 6-((iodoacetyl) amino)hexanoate (SIAX, 9-atom spacer), Succinimidyl6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)hexanoate (SIAXX, 16-atomspacer), and p-nitrophenyl iodoacetate (NPIA, 2-atom spacer). Oneordinarily skilled in the art also will recognize that a number of othercoupling agents or links, with different number of atoms, may be used.

Hydrophilic spacer atoms can be incorporated into linkers to increasethe distance between the reactive functional groups. For example,polyethylene glycol (PEG) can be incorporated into sulfo-GMBS.Hydrophilic molecules such as PEG have also been shown to decreasenon-specific binding (NSB) and increase hydrophilicity of surfaces whencovalently coupled. PEG can also be used as the primary linker material.

Free amine groups of components can also be attached to surfacemolecules or other components containing reactive amine groups viahomobifunctional linkers. Linkers such asdithiobis(succinimidylpropionate) (DSP, 8-atom spacer), disuccinimidylsuberate (DSS, 8-atom spacer), glutaraldehyde (4-atom spacer),Bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES, 9-atomspacer), all requiring high pH, can be used for this purpose. Examplesof homobifunctional sulfhydryl-reactive linkers include, but are notlimited to, 1,4-Di-[3″-2″-pyridyldithio)propion-amido]butane (DPDPB,16-atom spacer) and Bismaleimidohexane (BMH, 14-atom spacer). Forexample, these homobifunctional linkers are first reacted with athiolated surface in aqueous solution (for example PBS, pH 7.4), andthen in a second step, the thiolated antibody or protein is joined bythe link. Homo- and heteromultifunctional linkers can also be used.

Direct binding of components to thiol, amine, or carboxylic acidfunctional groups on surface molecules and other components be used toproduce compositions which exhibit viral binding (due to increaseddensity of components, for example), resulting in enhanced sensitivity.

As an example, when necessary to achieve high peptide coupling density,additional amino groups can be added to the surface molecules (such ascommercially obtained SPIO) as follows: First, to crosslink theparticles before the amination step, 3 ml of the colloid (10 mgFe/ml indouble-distilled water) was added to 5 ml of 5M NaOH and 2 ml ofepichlorohydrin (Sigma, St. Louis, Mo.). The mixture was agitated for 24hours at room temperature to promote interaction between the organicphase (epichlorohydrin) and aqueous phase (dextran-coated particlecolloid). In order to remove excess epichlorohydrin, the reacted mixturewas dialyzed against double-distilled water for 24 hours using adialysis cassette (10,000 Da cutoff, Pierce, Rockford Ill.). Aminogroups were added to the surface of the particles as follows: 0.02 ml ofconcentrated ammonium hydroxide (30%) was added to 1 ml of colloid(^(˜)10 mg Fe/ml). The mixture was agitated at room temperature for 24hours. The reacted mixture was dialyzed against double-distilled waterfor 24 hours. To further rinse the particles, the colloid was trapped ona MACS® Midi magnetic separation column (Miltenyi Biotec, AuburnCalif.), rinsed with PBS three times, and eluted from the column with 1ml PBS.

To conjugate peptides to SPIO, the particles can be re-suspended at aconcentration of 1 mg Fe/ml, and heterobifunctional linkerN-[a-maleimidoacetoxy]succinimide ester (AMAS; Pierce) can be added (2.5mg linker per 2 mg Fe) under vortexing. After incubation at roomtemperature for 40 minutes, the particles can be washed 3 times with 10ml PBS on a MACS column. The peptide with free terminal cysteine canthen be added (100 μg peptide per 2 mg Fe). After incubation overnightat 4° C., the particles can be washed again and re-suspended in PBS at aconcentration of 0.35 mg/ml of Fe. To quantify the number of peptidemolecules conjugated to the particles, a known amount of stock orAMAS-activated particles can be incubated with varying amounts of thepeptide. After completion of the incubation the particles can bepelleted at 100,000×g using Beckman TLA 100.3 ultracentrifuge rotor (30minutes) and the amount of the unbound peptide can be quantified byfluorescence. To cleave the conjugated peptide from the particles, theparticles can be incubated at 37° C. overnight at pH 10. Theconcentration of free peptide in the supernatant can then be determinedby reading fluorescence and by using the calibration curve obtained forthe same peptide. The fluorescence intensity of known amounts ofparticles can be plotted as a function of peptide conjugation density,and the slope equation can be used to determine conjugation density indifferent batches.

II.I. Pharmaceutical Compositions and Carriers

The disclosed compositions can be administered in vivo in apharmaceutically acceptable carrier. By “pharmaceutically acceptable” ismeant a material that is not biologically or otherwise undesirable,i.e., the material can be administered to a subject, along with the TT1Peptide composition, without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the pharmaceutical composition in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art. The materials can be insolution, suspension (for example, incorporated into microparticles,liposomes, and/or cells.

The TT1 Peptide compositions of the presently disclosed subject mattercan be used therapeutically in combination with one or morepharmaceutically acceptable carriers.

Suitable carriers and their formulations are described in Remington,1995. Typically, an appropriate amount of a pharmaceutically acceptablesalt is used in the formulation to render the formulation isotonic.Examples of the pharmaceutically acceptable carrier include, but are notlimited to, saline, Ringer's solution, and dextrose solution. The pH ofthe solution is in some embodiments from about 5 to about 8, and in someembodiments from about 7 to about 7.5. Further carriers includesustained release preparations such as semipermeable matrices of solidhydrophobic polymers containing the TT1 Peptide composition, whichmatrices are in the form of shaped articles, e.g., films, liposomes, ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers can be selected depending upon, for instance, theroute of administration and/or concentration of composition beingadministered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents, and the like, in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedTT1 Peptide compositions can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity, ortransdermally.

Preparations for parenteral administration can include sterile aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions, or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose, and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners, and the like can also be employed, as desired.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids, or binders can in some embodiments also be desirable.

Some of the compositions can be administered as a pharmaceuticallyacceptable acid- or base-addition salt, formed by reaction withinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, or phosphoric acidand organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,succinic acid, maleic acid, and fumaric acid, or by reaction with aninorganic base such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, and organic bases such as mono-, di-, trialkyl, and arylamines and substituted ethanolamines.

III. Combinatorial Chemistry/Screening Methods

The disclosed compositions can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. Alsodisclosed are the compositions that are identified through combinatorialtechniques or screening techniques in which the compositions comprisingSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or portions thereof, areused as the target in a combinatorial or screening protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties, such as interaction with gC1qR/p32. The moleculesidentified and isolated when using the disclosed compositions, such as aTT1 Peptide, are also disclosed. Thus, the products produced using thecombinatorial or screening approaches that involve the disclosedcompositions, such as a TT1 Peptide, are also considered hereindisclosed.

Disclosed herein are methods of screening for a compound that interactswith a gC1q/p32 receptor, comprising: bringing into contact a testcompound, a TT1 Peptide composition, and a gC1q receptor, wherein theTT1 Peptide composition comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO: 3; and detecting unbound TT1 Peptide composition, wherein a givenamount of unbound TT1 Peptide composition indicates a compound thatinteracts with gC1q/p32 receptor.

Also disclosed is a method of screening for a test compound thatmodulates gC1q/p32 receptor activity, comprising: contacting a cell thatcomprises the gC1q/p32 receptor with a test compound; and detectingaltered gC1q/p32 receptor activity; wherein altered levels of gC1q/p32receptor activity indicate a compound that modulates gC1q/p32 receptoractivity.

By “altered levels of activity” is meant that the gC1q/p32 receptor candisplay an increase or decrease in activity. The increase in activitycan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% increase, or a 1 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 75, or 100 fold or moreincrease in activity, as compared to a standard, control, or basallevel. The decrease in activity can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% decrease in activity as compared to a standard, control, or basallevel. For example, a test compound can interact with the gC1q/p32receptor in such a way as to decrease the ability of the gC1q/p32receptor to interact with another compound, thereby decreasing itsactivity. In another example, a test compound can prevent the synthesisof the gC1q/p32 receptor, thereby decreasing its activity in that way.

Disclosed is a method of screening for a test compound that interactswith the gC1q/p32 receptor, comprising contacting a cell that comprisesthe gC1q/p32 receptor with a test compound; and detecting interactionbetween the gC1q/p32 receptor and the test compound. After the testcompound has been shown to interact with the gC1q/p32 receptor, it canfurther be tested for its ability to modulate gC1q/p32 receptoractivity, including the ability to treat a gC1q/p32 receptor-relateddisorder.

Further disclosed is a method of screening for a test compound that canbe used to treat a gC1q/p32 receptor-related disorder, such as cancer,comprising: contacting a cell that comprises the gC1q/p32 receptor witha test compound; and detecting altered gC1q/p32 receptor activity;wherein altered levels of gC1q/p32 receptor activity indicate a compoundthat can modulate gC1q/p32 receptor activity. After the test compoundhas been shown to modulate gC1q/p32 receptor activity, the test compoundcan then be tested for its ability to treat a gC1q/p32 receptor-relateddisorder.

The modulation can comprise a decrease in gC1q/p32 receptor activity,expression, or the ability to treat a gC1q/p32 receptor-related disease.By a “decrease” is meant that the activity is less in the presence ofthe test compound than not in the presence of the test compound. Themodulation can comprise an increase in gC1q/p32 receptor activity orrelated activity. By an “increase” is meant that the activity is greaterin the presence of the test compound than not in the presence of thetest compound.

The response of the gC1q/p32 receptor can be measured in the presence ofvarious concentrations of test compound. The measuring steps can alsocomprise measuring the response at various concentrations of the testcompound. For example, the concentration of the test compound can rangefrom 1 nM to 1000 μM.

Assays contemplated by the invention include both binding assays andactivity assays; these assays may be performed in conventional or highthroughput formats. Modulator screens are designed to identifystimulatory and inhibitory agents. The sources for potential agents tobe screened include natural sources, such as a cell extract (e.g.,invertebrate cells including, but not limited to, bacterial, fungal,algal, and plant cells) and synthetic sources, such as chemical compoundlibraries or biological libraries such as antibody substance or peptidelibraries. Agents are screened for the ability to either stimulate orinhibit the activity. Binding assays are used to detect activity levels.Both functional and binding assays of activity are readily adapted toscreens for modulators such as agonist (stimulatory) and antagonist(inhibitory) compounds.

Contemplated herein are a multitude of assays to screen and identifymodulators, such as agonists and antagonists, of the gC1q/p32 receptor(and downstream activity). In some embodiments, the cell is immobilizedand interaction with a candidate modulator is detected. In someembodiments, the test compound is immobilized. In yet another example,interaction between gC1q/p32 receptor and the test compound is assessedin a solution assay. Another contemplated assay involves a variation ofthe di-hybrid assay wherein a modulator of protein/protein interactionsis identified by detection of a positive signal in a transformed ortransfected host cell.

Candidate modulators for screening according to contemplated by theinvention include any chemical compounds, including libraries ofchemical compounds. There are a number of different libraries used forthe identification of small molecule modulators, including: (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules. Chemical libraries consist of random chemical structures, oranalogs of known compounds, or analogs of compounds that have beenidentified as “hits” or “leads” in prior drug discovery screens, some ofwhich may be derived from natural products or from non-directedsynthetic organic chemistry. Natural product libraries are collectionsof microorganisms, animals, plants, or marine organisms which are usedto create mixtures for screening by: (1) fermentation and extraction ofbroths from soil, plant or marine microorganisms or (2) extraction ofplants or marine organisms. Natural product libraries includepolyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) thereof. For a review, see e.g., Cane et al., 1998.Combinatorial libraries are composed of large numbers of peptides,oligonucleotides, or organic compounds as a mixture. These libraries arerelatively easy to prepare by traditional automated synthesis methods,PCR, cloning, or synthetic methods. of particular interest arenon-peptide combinatorial libraries. Still other libraries of interestinclude peptide, protein, peptidomimetic, multiparallel syntheticcollection, recombinatorial, and polypeptide libraries. For a review ofcombinatorial chemistry and libraries created therefrom, see Myers,1997. Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to modulate activity.

Candidate modulators contemplated by the invention can be designed andinclude soluble forms of binding partners, as well as chimeric, orfusion, proteins thereof. A “binding partner” as used herein broadlyencompasses non-peptide modulators, peptide modulators (e.g.,neuropeptide variants), antibodies (including monoclonal and polyclonalantibodies, single chain antibodies, chimeric antibodies,bifunctional/bispecific antibodies, humanized antibodies, humanantibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR and/or antigen-bindingsequences, which specifically recognize a polypeptide as disclosedherein), antibody fragments, and modified compounds comprising antibodydomains that are immunospecific for the expression product.

Assays that measure binding or interaction of compounds with targetproteins include assays that identify compounds that inhibit unfoldingor denaturation of a target protein, assays that separate compounds thatbind to target proteins through affinity ultrafiltration followed by ionspray mass spectroscopy/HPLC methods or other physical and analyticalmethods, capillary electrophoresis assays and two-hybrid assays.

One such screening method to identify direct binding of test ligands toa target protein is described in U.S. Pat. No. 5,585,277, incorporatedherein by reference. This method relies on the principle that proteinsgenerally exist as a mixture of folded and unfolded states, andcontinually alternate between the two states. When a test ligand bindsto the folded form of a target protein (i.e., when the test ligand is aligand of the target protein), the target protein molecule bound by theligand remains in its folded state. Thus, the folded target protein ispresent to a greater extent in the presence of a test ligand which bindsthe target protein, than in the absence of a ligand. Binding of theligand to the target protein can be determined by any method whichdistinguishes between the folded and unfolded states of the targetprotein. The function of the target protein need not be known in orderfor this assay to be performed. Virtually any agent can be assessed bythis method as a test ligand, including, but not limited to, metals,polypeptides, proteins, lipids, polysaccharides, polynucleotides andsmall organic molecules.

Another method for identifying ligands of a target protein is describedin Wieboldt et al., 1997, incorporated herein by reference. Thistechnique screens combinatorial libraries of 20-30 agents at a time insolution phase for binding to the target protein. Agents that bind tothe target protein are separated from other library components by simplemembrane washing. The specifically selected molecules that are retainedon the filter are subsequently liberated from the target protein andanalyzed by HPLC and pneumatically assisted electrospray (ion spray)ionization mass spectroscopy. This procedure selects library componentswith the greatest affinity for the target protein, and is particularlyuseful for small molecule libraries.

Alternatively, such binding interactions are evaluated indirectly usingthe yeast two-hybrid system described in Fields et al., 1989 and Fieldset al., 1994, both of which are incorporated herein by reference. Thetwo-hybrid system is a genetic assay for detecting interactions betweentwo proteins or polypeptides. It can be used to identify proteins thatbind to a known protein of interest, or to delineate domains or residuescritical for an interaction. Variations on this methodology have beendeveloped to clone genes that encode DNA binding proteins, to identifypeptides that bind to a protein, and to screen for drugs. The two-hybridsystem exploits the ability of a pair of interacting proteins to bring atranscription activation domain into close proximity with a DNA bindingdomain that binds to an upstream activation sequence (UAS) of a reportergene, and is generally performed in yeast. The assay requires theconstruction of two hybrid genes encoding (1) a DNA-binding domain thatis fused to a first protein and (2) an activation domain fused to asecond protein. The DNA-binding domain targets the first hybrid proteinto the UAS of the reporter gene; however, because most proteins lack anactivation domain, this DNA-binding hybrid protein does not activatetranscription of the reporter gene. The second hybrid protein, whichcontains the activation domain, cannot by itself activate expression ofthe reporter gene because it does not bind the UAS. However, when bothhybrid proteins are present, the noncovalent interaction of the firstand second proteins tethers the activation domain to the UAS, activatingtranscription of the reporter gene.

The literature is replete with examples of the use of radiolabeledligands in HTS binding assays for drug discovery (see Williams, 1991 andSweetnam et al., 1993, each of which is herein incorporated by referencein its entirety for their teaching concerning high throughput screens).It is also possible to screen for novel neuroregeneration compounds withradiolabeled ligands in HTS binding screens. Other reasons thatrecombinant receptors are preferred for HTS binding assays includebetter specificity (higher relative purity) and ability to generatelarge amounts of receptor material (see Hodgson, 1992).

A variety of heterologous systems are available for expression ofrecombinant proteins and are well known to those skilled in the art.Such systems include bacteria (Strosberg et al., 1992), yeast (Pausch,1997), several kinds of insect cells (Vanden Broeck, 1996), amphibiancells (Jayawickreme et al., 1997), and several mammalian cell lines(CHO, HEK293, COS, etc.; see Gerhardt et al., 1997; Wilson et al.,1998). These examples do not preclude the use of other possible cellexpression systems, including cell lines obtained from nematodes (PCTInternational Patent Application Publication No. WO 98/37177).

Inhibition of gC1qR/p32, or downstream products or genes relatedthereto, can result in a variety of biological responses, which aretypically mediated by proteins expressed in the host cells. The proteinscan be native constituents of the host cell or can be introduced throughwell-known recombinant technology. They can be mutants of nativevarieties as well. The proteins can be intact or chimeric.

Fluorescence changes can also be used to monitor ligand-induced changesin membrane potential or intracellular pH; an automated system suitablefor HTS has been described for these purposes (Schroeder et al., 1996).Among the modulators that can be identified by these assays are naturalligand compounds; synthetic analogs and derivatives of natural ligands;antibodies, antibody fragments, and/or antibody-like compounds derivedfrom natural antibodies or from antibody-like combinatorial libraries;and/or synthetic compounds identified by high throughput screening oflibraries; and other libraries known in the art. All modulators thatinteract with gC1qR/p32 are useful for identifying TT1 Peptide-likepolypeptides (e.g., for diagnostic purposes, pathological purposes, andother purposes known in the art). Agonist and antagonist modulators areuseful for up-regulating and down-regulating gC1qR/p32 activity,respectively, for purposes described herein.

The assays may be performed using single putative modulators; they mayalso be performed using a known agonist in combination with candidateantagonists (or vice versa). Detectable molecules that may be usedinclude, but are not limited to, molecules that are detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,radioactive, and optical means, including but not limited tobioluminescence, phosphorescence, and fluorescence. These detectablemolecules should be a biologically compatible molecule and should notcompromise the biological function of the molecule and must notcompromise the ability of the detectable molecule to be detected.Exemplary detectable molecules are optically detectable molecules,including optically detectable proteins, such that they may be excitedchemically, mechanically, electrically, or radioactively to emitfluorescence, phosphorescence, or bioluminescence. Other exemplarydetectable molecules are inherently fluorescent molecules, such asfluorescent proteins, including, for example, Green Fluorescent Protein(GFP). The detectable molecule can be conjugated to the composition bymethods as described in U.S. Pat. Nos. 5,891,646 and 6,110,693, both toBarak et al.). The detectable molecule can be conjugated at thefront-end, at the back-end, or in the middle.

IV. Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation or the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions, such as a TT1Peptide, are also disclosed. Thus, the products produced using themolecular modeling approaches that involve the disclosed compositions,such as a TT1 Peptide, are also considered herein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This can be achieved through structuralinformation and computer modeling.

Computer modeling technology allows visualization of thethree-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass., United States ofAmerica). CHARMm performs the energy minimization and molecular dynamicsfunctions. QUANTA performs the construction, graphic modeling andanalysis of molecular structure. QUANTA allows interactive construction,modification, visualization, and analysis of the behavior of moleculeswith each other.

A number of publications review computer modeling of drugs interactivewith specific proteins, such as Rotivinen et al., 1988; Ripka, 1988;McKinaly & Rossmann, 1989; Perry & Davies, 1989; Lewis & Dean, 1989;and, with respect to a model enzyme for nucleic acid components, Askewet al., 1989. Other computer programs that screen and graphically depictchemicals are available from companies such as BioDesign, Inc.,Pasadena, Calif., United States of America; Allelix, Inc., Mississauga,Ontario, Canada; and Hypercube, Inc., Cambridge, Ontario, Canada.Although these are primarily designed for application to drugs specificto particular proteins, they can be adapted to design of moleculesspecifically interacting with specific regions of DNA or RNA, once thatregion is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

V. Compositions with Similar Functions

It is understood that the compositions disclosed herein have certainfunctions, such as interacting with gC1qR/p32. Disclosed herein arecertain structural requirements for performing the disclosed functions,and it is understood that there are a variety of structures which canperform the same function which are related to the disclosed structures,and that these structures will ultimately achieve the same result, forexample stimulation or inhibition.

VI. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include a TT1 Peptide and/orgC1q/p32 receptors.

VII. Mixtures

Whenever A method for the presently disclosed subject matter involvesmixing or bringing into contact compositions or components or reagents,performing the method creates a number of different mixtures. Forexample, if the method includes 3 mixing steps, after each one of thesesteps a unique mixture is formed if the steps are performed separately.In addition, a mixture is formed at the completion of all of the stepsregardless of how the steps were performed. The present disclosurecontemplates these mixtures, obtained by the performance of thedisclosed methods as well as mixtures containing any disclosed reagent,composition, or component, for example, disclosed herein.

VIII. Systems

Disclosed are systems useful for performing, or aiding in theperformance of, the disclosed method. Systems generally comprisecombinations of articles of manufacture such as structures, machines,devices, and the like, and compositions, compounds, materials, and thelike. Such combinations that are disclosed or that are apparent from thedisclosure are contemplated.

IX. Computer Readable Media

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understand what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

X. Peptide Synthesis

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

One method of producing the disclosed proteins, such as a TT1 Peptide ora conjugate thereof, is to link two or more peptides or polypeptidestogether by protein chemistry techniques. For example, peptides orpolypeptides can be chemically synthesized using currently availablelaboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) orBoc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,Foster City, Calif., United States of America). One skilled in the artcan readily appreciate that a peptide or polypeptide corresponding tothe disclosed proteins, for example, can be synthesized by standardchemical reactions. For example, a peptide or polypeptide can besynthesized and not cleaved from its synthesis resin whereas the otherfragment of a peptide or protein can be synthesized and subsequentlycleaved from the resin, thereby exposing a terminal group which isfunctionally blocked on the other fragment. The peptide or polypeptideis independently synthesized in vivo as described herein. Once isolated,these independent peptides or polypeptides can be linked to form apeptide or fragment thereof via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides, or whole protein domains (Abrahmsen etal., 1991). Alternatively, native chemical ligation of syntheticpeptides can be utilized to synthetically construct large peptides orpolypeptides from shorter peptide fragments. This method consists of atwo step chemical reaction (Dawson et al., 1994). The first step is thechemoselective reaction of an unprotected synthetic peptide-thioesterwith another unprotected peptide segment containing an amino-terminalCys residue to give a thioester-linked intermediate as the initialcovalent product. Without a change in the reaction conditions, thisintermediate undergoes spontaneous, rapid intramolecular reaction toform a native peptide bond at the ligation site (Baggiolini al., 1992;Clark-Lewis et al., 1994; Clark-Lewis et al., 1991; Rajarathnam et al.,1994).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer et al., 1992).This technique has been used to synthesize analogs of protein domains aswell as large amounts of relatively pure proteins with full biologicalactivity (deLisle et al., 1992).

XI. Methods

Disclosed herein are methods of interacting compositions with gC1qR/p32.Such interactions can be, for example, selective, targeted, or homing.Interaction with gC1qR/p32 can be mediated by a TT1 Peptide and caninvolve any TT1 Peptide or TT1 peptide composition as described herein.Interaction with gC1qR/p32 can be useful for detecting and/or treatingdiseases and conditions, such as diseases and/or conditions associatedwith gC1qR/p32.

Disclosed herein are methods of treating a disease associated withgC1q/p32 receptor comprising identifying a subject having a diseaseassociated with the gC1q/p32 receptor; and administering to the subjecta composition comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3(i.e., a TT1 Peptide).

Also disclosed are methods of treating a disease associated withgC1q/p32 receptor comprising identifying a subject having a diseaseassociated with the gC1q/p32 receptor; and administering to the subjecta composition that interacts with the gC1q/p32 receptor in the samelocation as a TT1 Peptide, thereby treating a disease associated withthe gC1q/p32 receptor. The composition that interacts with the gC1q/p32receptor can be, for example, an antibody, protein, or chemical.

Disclosed are methods of delivering a TT1 Peptide composition to agC1q/p32 receptor, wherein the TT1 Peptide composition comprises amoiety linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3; wherein the method comprises bringing into contact the TT1Peptide composition and a cell, thereby delivering the TT1 Peptidecomposition to the gC1q/p32 receptor.

In some embodiments, the cell is in a subject. When the cell is in asubject, the cell can be selected for its potential to comprise agC1q/p32 receptor by detecting the presence of gC1q/p32 receptor onanother cell of the subject.

Also disclosed are methods of delivering a TT1 Peptide composition to agC1q/p32 receptor, wherein the TT1 Peptide composition comprises amoiety linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3; comprising: selecting a cell for its potential to comprisea gC1q/p32 receptor; and bringing into contact the TT1 Peptidecomposition and the cell, thereby delivering the TT1 Peptide compositionto the gC1q/p32 receptor.

Also disclosed are methods of detecting interaction between a gC1q/p32receptor and a TT1 Peptide composition, wherein the TT1 Peptidecomposition comprises a moiety linked to a composition comprising SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, the method comprising: selecting acell for its potential to comprise a gC1q/p32 receptor; bringing intocontact the TT1 Peptide composition and the cell; and detectinginteraction between the gC1q/p32 receptor and the TT1 Peptidecomposition.

Disclosed are methods of determining and/or assessing gC1q/p32 receptorlevel in a cell of a subject, comprising: bringing into contact a cellof the subject and a TT1 Peptide composition comprising a detectableagent linked to a composition comprising SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3; and detecting the level of TT1 Peptide compositioninteracting with gC1q/p32 receptor, thereby determining and/or assessinggC1q/p32 receptor level in the cell. The level of gC1q/p32 receptor inthe subject is compared to a previous measurement in the same subject,or can be compared to a control level or standard level.

Also disclosed are methods of identifying a subject having a diseaseassociated with gC1q/p32 receptor, the method comprising bringing intocontact a cell of the subject and a TT1 Peptide composition, wherein theTT1 Peptide composition comprises a moiety linked to a compositioncomprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and detectinginteraction between gC1q/p32 receptor and the TT1 Peptide composition,thereby detecting the presence or level of gC1q/p32 on the cell, whereinthe presence or level of gC1q/p32 receptor on the cell identifies thesubject as having a disease associated with a gC1q/p32 receptor.

Also disclosed are methods of screening for a compound that interactswith a gC1q/p32 receptor, comprising bringing into contact a testcompound, a TT1 Peptide composition, and a gC1q/p32 receptor, whereinthe TT1 Peptide composition comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3; and detecting unbound TT1 Peptide composition, wherein a givenamount of unbound TT1 Peptide composition indicates a composition thatinteracts with gC1q/p32 receptor. The TT1 Peptide composition cancomprise a moiety, wherein the moiety comprises SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3. In some embodiments, the moiety can be a detectableagent. Methods of screening are discussed in more detail below.

Further disclosed herein is a method of treating or preventing a diseasein a subject associated with gC1q/p32 receptor, the method comprisingadministering to the subject a composition that modulates gC1q/p32receptor expression or activity, thereby treating a disease in a subjectassociated with the gC1q/p32 receptor. The subject can have cancer. Thecomposition can have a therapeutic effect on the cancer. The size of atumor can be reduced. The growth of a tumor can be reduced, stopped orreversed.

Expression or activity of the gC1q/p32 receptor can be inhibited. Thiscan occur by the use of interfering nucleic acid, such as shRNA orsiRNA. Activity of the gC1q/p32 receptor can be inhibited by a TT1Peptide, an antibody, or a small molecule mimic of a TT1 Peptide. Themethods of treating or preventing cancer disclosed herein can be used inconjunction with other treatment therapies as well.

The therapeutic effect of the compositions disclosed herein can be aslowing in the increase of or a reduction of tumor burden. This slowingin the increase of, or reduction in the tumor burden, can be 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%,800%, ⁹⁰⁰%, or 1000% or more improvement in the increase of, orreduction in the tumor burden of, compared with a non-treated tumor, ora tumor treated by a different method.

The gC1q/p32 receptor involved in the disclosed methods can be, forexample, on or in a cell. The cell can be in any context, such as in anorganism, in situ, ex vivo, in culture, and/or in vitro.

The disclosed compositions can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers. Anon-limiting list of different types of cancers can be as follows:lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomasof solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas,gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas,histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas,AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers ingeneral.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,or pancreatic cancer.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the presently disclosed subject matter, and are notintended to limit the scope of what the inventors regard as theirinvention nor are they intended to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Celsius, and pressure is at or near atmospheric.

Example 1 Purification and Oligomerization Analysis of the p32 Proteinand Biopanning

6×-His-Tagged P32 was expressed in Rosetta-gami-2 cells (Novagen). Theprotein was purified by IMAC using HIS-select resin (Sigma) with animidizole gradient from 20-300 mM and eluted fractions were analyzed onanalytical SDS-PAGE (see FIG. 1A). A sedimentation velocity assay thatallowed determination of the multimeric state of proteins was performed,and the results are summarized in FIG. 1B and in Table 4. As showntherein, the sedimentation velocity assay demonstrated that similarly tothe native p32 protein, recombinant p32 is present predominantly (>97%)as a trimer with sedimentation coefficient of 5.447 and apparentmolecular weight for the complex of 79.2 kDa (see FIG. 1B).

TABLE 4 Sedimentation Velocity Assay Results Sample S S(20 w) % ApparentMw 1 2.117 2.201 2.7 19.2 kDa 2 53447 5.663 97.3 79.2 kDa

Milligram quantities of recombinant His-tagged p32 protein wereexpressed in E. coli and coated onto Ni-NTA magnetic beads, andbiopanning was performed using two T7 bacteriophage peptide libraries(cyclic Cx7C and linear X7 library). After three rounds of selection, a500-800 fold increase in binding to recombinant p32 was seen for bothlibraries (see FIG. 2A).

Variant peptide-encoding genomic DNA of phage was sequenced and analyzedfor the presence of consensus motifs. After two rounds of selection,both linear and cyclic libraries converged to contain an RGXRS (SEQ IDNO: 4) pentapeptide motif (see FIG. 2B). The binding of RGXRS (SEQ IDNO: 4)-displaying phage to p32 was specific, as these phage bound tomagnetic beads coated with p32 protein and not to beads coated withligand-binding b1b2 domain of the cell and tissue penetration receptorNRP-1 or BSA (see FIG. 3).

An exemplary RGXRS (SEQ ID NO: 4) pentapeptide motif-containing peptidewith the sequence CKRGARSTC (SEQ ID NO: 3) was designated as the “TT1Peptide” and used it for further studies. Mutating any amino acid in theconsensus motif of TT1 abolished the phage binding to p32. This newpeptide conferred about 15-fold stronger phage binding to immobilizedp32 protein than did the canonical LyP-1 peptide. Fluorescencepolarization assays with the synthetic peptides confirmed that the TT1Peptide binding to p32 was specific and that the TT1 Peptide shared thebinding site on p32 with the LyP-1 peptide (see FIGS. 4 and 5).

Example 2 In Vitro Internalization and In Vivo Homing of the TT1 Peptide

The ability of the TT1 Peptide to bind to cultured breast tumor cellswas tested in vitro. Exemplary results are shown in FIG. 6. It appearedthat the synthetic fluorophore-labeled TT1 Peptide rapidly bound to thesurface of 4T1 and MCF10 breast cancer cells and was internalized overtime.

Systemically administered LyP-1 and iRGD tumor-penetrating peptidesefficiently home to and penetrate orthotopic breast tumors in vivo.Intravenously administered TT1 Peptide had similar activity, and ithomed to breast tumors in mice bearing 4T1 syngeneic (see FIG. 7A)tumors, as well as MCF7 (see FIG. 7B) and MDA-MB-231 xenografts.

In a different model, the TT1 Peptide homed to and penetratedatherosclerotic lesions in ApoE null mice maintained on high-fat diet.The homing appeared to be more robust than seen with the LyP-1 peptide(see FIG. 8).

Example 3 The Cryptic CendR Element in the TT1 Peptide can be Activatedby Proteolytic Cleavage

The cryptic (internal) R/KXXR/K C-end Rule (CendR) element is the keydeterminant of the ability of tumor-penetrating and tissue-penetratingpeptides to reach deep into parenchymal extravascular tumor tissue. Inthese peptides, the CendR motif is exposed at the C-terminus byendogenous cell-surface proteases to trigger interaction of the peptidewith cell penetration and tissue penetration receptor NRP-1.Upregulation of extracellular proteolysis machinery is one of thehallmarks common to tumors and atherosclerotic lesions. Urokinase-typeplasminogen activator (uPA) and matrix metalloproteinases are thought tobe of central importance for tumor invasion and metastasis, andextracellular matrix remodeling.

The p32 binding motif with consensus sequence RGXRS (SEQ ID NO: 4; i.e.,the RGXRS (SEQ ID NO: 4) pentapeptide motif) contains a cryptic RGXRCendR element that can be unmasked by a tryptic cleavage. Indeed,exposure of the TT1 Peptide to trypsin resulted in affinity switchingfrom p32 to NRP-1 b1b2 (see FIG. 9). In addition, exposure of TT phageto recombinant urokinase-type plasminogen activator (uPA) led to180-fold enhancement of the NRP-1 b1b2 binding.

Thus, the TT1 Peptide had a dual, protelytically switchable targetspecificity. The peptide was first recruited to the p32 protein presenton the surface of tumor macrophages, tumor lymphatic cells, and cancercells in hypoxic areas of tumors, followed by its processing by uPA (andpotentially other proteases) in the tumor extracellular milieu.Processed peptide with free C-terminal CendR motif bound to cell andtissue penetration receptor NRP-1 to trigger tumor penetration ofconjugated and co-administered payloads. As both determinants of the TT1Peptide's specificity, p32 and proteases like uPA, are expressedpredominantly in tumor tissue, the generation of the CendR fragmentcapable of triggering cell- and tissue-penetration appeared to belimited to tumors. A similar process likely accounts for the homing andpenetration of the TT1 Peptide in atherosclerotic plaques.

Example 4 Binding of the RGXR(S/T) (SEQ ID NOs: 4 and 5) Motif to p32 isConformation and Position Independent

The cyclic structure of the LyP-1 Peptide is required for its binding top32 and for its biological activities. In contrast, the p32-bindingRGARS (SEQ ID NO: 4) motif of the TT1 Peptide did not require the motifto be in a cyclic context. A TT1 Peptide variant with cysteine residuesreplaced by alanine residues retains the binding activity (see FIG. 10).Of note, it is possible that in vivo, the cyclic structure of the TT1Peptide could be beneficial and increase the stability of the peptide.Importantly, the p32 binding activity of the RGARS (SEQ ID NO: 4) motifwas not adversely affected by flanking GGSG (SEQ ID NO: 16) linkers.These features render the TT1 family of peptides particularly suitablefor design of modular targeting peptides.

Materials and Methods for EXAMPLES 5-7

Peptide and NW Conjugation.

Peptides were synthesized with an automatic microwave-assisted peptidesynthesizer (Liberty; CEM) using standard solid-phase Fmoc/tBu chemistrywith 2-(1H-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (AnaSpec) as the coupling reagent. An extracysteine residue was added to the N-terminus of the cyclic peptides(LyP-1 and cTT1) as described (Sugahara et al., 2009). During thesynthesis, the peptides were labeled with 5(6)-carboxyfluorescein (FAM;Sigma-Aldrich) with a 6-aminohexanoic acid spacer to separate the dyefrom the sequence. The peptides were cleaved from the resin with 95%trifluoroacetic acid (Sigma-Aldrich) with 2.5% water andtriisopropylsilane (Sigma-Aldrich). Subsequent purification byhigh-performance liquid chromatography (Gilson) yielded peptideswith >90% purity. For NW coupling, aminated NWs were pegylated withmaleimide-5KPEG-NHS (JenKem Technology) and peptides were conjugated tothe nanoparticles through a thioether bond between the cysteine thiol inthe peptide and the maleimide on the functionalized particles.

Cell Lines and Tumors.

MCF10CA1a human tumor cells were obtained from the Barbara Ann KarmanosCancer Institute (Detroit, Mich.). Cells were maintained in Dulbecco'sModified Eagle Medium supplemented with 10% fetal bovine serum, 1%Glutamine-Pen-Strep and 100 ng/ml of human epidermal growth factor(Sigma-Aldrich, St. Louis, Mo., United States of America).

To produce MCF10CA1a tumors, athymic nude mice were orthotopicallyinjected into the mammary fat pad with 2×10⁶ cells suspended in 100 μlof PBS. Animal experimentation was performed according to the proceduresapproved by the Animal Research Committee at the Sanford-Burnham MedicalResearch Institute.

In Vivo Peptide Homing.

Orthotopic breast tumor xenografts were used when they reached 0.5-1 cmin size. NWs conjugated peptides were injected into the tail vein (7.5mg of iron per kilogram of body weight). After 5-6 hours of NWcirculation the mice were euthanized by cardiac perfusion with PBS underanesthesia, and tumors and organs were dissected and analyzed forparticle homing.

Immunofluorescence.

Tissues from mice injected with nanoparticles were fixed in 4%paraformaldehyde overnight at 4° C., cryoprotected in 30% sucroseovernight, and frozen in optimal cutting temperature (OCT) embeddingmedium. Subsequently, 5-7 m sections were cut and stored at −20° C.until used. For immunostaining, tissue sections were first incubated for1 hour at room temperature with 10% serum from the species in which thesecondary antibody was generated, followed by incubation with theprimary antibody overnight at 4° C. Rat monoclonal anti-mouse CD31 (10μg/mL) was from BD Pharmingen). The primary antibody was detected withAlexa 594-labeled goat anti-rat secondary antibody (1:1000; MolecularProbes). Each staining experiment included sections stained only withsecondary antibodies as negative controls. Nuclei were stained with DAPI(4′,6-diamidino-2-phenylindole; 5 μg/mL; Molecular Probes). The sectionswere mounted in gel/mount mounting medium (Biomeda) and viewed under aFluoView 500 confocal microscope (Olympus America; 200 micronmicrographs, 20 m magnification and 50 microns, 40 μm).

Example 5 Peptide and Nanoparticle Synthesis

Potential p32-binding peptides were synthesized as free peptides and aschimeras with _(D)(KLAKLAK)₂ (SEQ ID NO: 42). These included LyP-1, anexemplary cyclic TT1 Peptide (CycTT1), and an exemplary linear TT1Peptide (LinTT1). The amino acid sequences of these peptides are shownin Table 5. The peptides were also labeled with a fluorescein (FAM)group. All peptides were successfully synthesized at >90% purity, andthe structure of each peptide was confirmed by mass spectrometryanalysis. The peptides were covalently conjugated onto aminogroup-functionalized dextran-coated superparamagnetic iron oxidenanoworms (NWs) as such or as the homing moiety in a chimeric peptidewith the _(D)(KLAKLAK)₂ (SEQ ID NO: 42) drug peptide (see FIG. 11) fortesting. Fluorescence measurement was used to compare the rate ofpeptide coupling. All peptides coupled at approximately the sameefficiency.

TABLE 5 Amino acid Sequences of Exemplary p32-binding Peptides FullLength Amino Acid Sequence CGKRK (SEQ ID NO: 6) AKRGARSTA (LinTT1; (SEQID NO: 2) CKRGARSTC (CycTT1; SEQ ID NO: 3) CGNKRTRGC (LyP-1; SEQ ID NO:7) CGKRK-_(D)(KLAKLAK)₂ (SEQ ID NO: 8) LyP1-_(D)(KLAKLAK)₂ (SEQ ID NO:9) LinTT1-_(D)(KLAKLAK)₂ (SEQ ID NO: 10)

Example 6 Tumor Homing and Tissue Penetration

The peptides were assayed for tumor homing to allow selection of thepeptide with optimal homing and penetration properties on the NWs and inthe context of the homing peptide-_(D)(KLAKLAK)₂—NW nanosystem. Mouseglioblastomas were used initially to show the efficacy of theCGKRK-_(D)(KLAKLAK)₂ (SEQ ID NO: 8) NWs. More recently, breast cancerswere shown to respond to this nanosystem the same way as glioblastomas(see Agemy et al., 2013). This finding made it possible to use theorthotopic tumors (in the mammary fat pad), which are technically easierto produce than orthotopic glioblastomas.

Tumor homing of peptide-NWs was then examined. Homing experiments showedthat the new peptides were more effective in causing tumor homing of NWsthan CGKRK (SEQ ID NO: 6). These included the TT1 Peptide, a crypticCendR motif-containing p32-binding peptide. The TT1 Peptide was selectedby phage library screening for binding to p32 as described herein above.A cyclic version of the TT1 Peptide had a high affinity for p32—higherthan that of the LyP-1 Peptide or of a CGKRK (SEQ ID NO: 6) Peptide—butits low-affinity linear variant turned out to be the most effectivepeptide in the nanoparticle context (see FIG. 12).

The cyclic TT1 Peptide tended to cause aggregation of the peptide-coatednanoparticles, which might explain the low activity of this peptide. Themultivalent presentation of the linear TT1 Peptide likely made up forits low affinity for the receptor. A summary of the quantifiedpeptide-NW homing results in shown in FIG. 13. The linear TT1 Peptidewas selected for further study as a _(D)[KLAKLAK]₂ (SEQ ID NO: 42)conjugate on NWs. The CGKRK (SEQ ID NO: 6) and LyP-1 Peptides wereincluded for comparison.

Example 7 Tumor Homing of Homing Peptide-_(D)[KLAKLAK]₂ (SEQ ID NO: 4)NWs

CGKRK-_(D)[KLAKLAK]₂ (SEQ ID NO: 8) NWs have been shown previously toaccumulate in tumor blood vessels without penetrating into theextravascular tumor tissue Agemy et al., 2011; 2013. The LyP-1 Peptidewas more effective than the CGKRK (SEQ ID NO: 6) Peptide, but the linearTT1-_(D)[KLAKLAK]₂ (SEQ ID NO: 10) NWs were most effective inpenetrating outside tumor vessels among the peptides tested (see FIG.14). Quantification of the NW fluorescence in the tumors for thepeptides tested is shown in FIG. 15.

Example 8 Tumor Growth Suppression Using TT1 Peptide-ConjugatedNanoworms

Tumors were induced by orthotopically inoculating immunocompromised nudemice with human MCF10-CA1a breast cancer cells. Treatment was stated 3-4weeks after the inoculation when the tumor volume was of about 50 mm3.The mice received vehicle only (PBS; 5 mice), linear TT1-NWs without thedrug peptide (5 mice), CGKRK-_(D)(KLAKLAK)₂ (SEQ ID NO: 8) NWs used inprevious work (see Agemy et al., 2011; 2013; 5 mice), or linearTT1-_(D)(KLAKLAK)₂ (SEQ ID NO: 10) NWs (8 mice). The linear TT1 Peptideportion of the TT1-NW conjugate had the structureFAM-X-Cys-X-AKARGARSTSA (SEQ ID NO: 2)-AMIDE, where X is aminohexanoicacid. The linear TT1 Peptide portion of the linear TT1-_(D)(KLAKLAK)₂—NWconjugate had the structure FAM-X-KLAKLAKKLAKLAK (SEQ ID NO: 42)X-Cys-X-AKARGARSTSA (SEQ ID NO: 2)-AMIDE, where X is aminohexanoic acid.

The results are presented in FIG. 16. As shown in FIG. 16, astatistically significant difference between the PBS control and all 3treatment groups was observed. The linear TT1-_(D)(KLAKLAK)₂ (SEQ ID NO:10) NW group was not statistically different from the other twotreatments groups. However, this group had two long-term survivors (25%)that appeared to be complete cures. The linear TT1-coated NWs alone hada substantial effect on tumor growth. This effect is in agreement withthe anti-tumor effect described for the LyP-1 peptide (Laakkonen et al.,2004). However, the linear TT1-NWs appeared to be more effective thanthe soluble LyP-1 peptide described in Laakkonen et al., 2004. While notwishing to be bound by any particular theory of operation, it ispossible that relative to the LyP-1 Peptide conjugates described inLaakkonen et al., 2004, the presently disclosed linearTT1-_(D)(KLAKLAK)₂ (SEQ ID NO: 10) NW were characterized by a longerblood half-life and/or more efficient processing to theneuropilin-1-binding CendR fragment.

Example 9 Tumor Treatment with LinTT1-_(D)[KLAKLAK]₂ Micelles in aMCF10CA1a Breast Cancer Model

Tumors were induced by orthotopically inoculating immunocompromised nudemice with human MCF10-CA1a breast cancer cells. Treatment was started2-3 weeks after the inoculation when the tumor volume was of about 40-50mm³. The mice received vehicle only (PBS; 5 mice) orTT1-_(D)(KLAKLAK)₂-micelles (280 μg of micelles/mouse, 5 mice; everyother day for 3 weeks. The results are presented in FIG. 17.

The data presented in FIG. 17 showed a statistically significantdifference between the PBS control and micelle treatment groups by oneway ANOVA (Tukey's posthoc test, n=3 mice per group).

Discussion of the Examples

Disclosed herein is an advanced theranostic nanosystem to home activeagents and other cargo to tumors. The results disclosed herein show thata new p-32-binding CendR motif peptide, referred to herein as the TT1Peptide or the TT1 Family of Peptides, was the most effectivetumor-homing peptide when tested coated onto nanoparticles, and in thecontext of the complete nanosystem. The TT1 Peptide-based nanosystem waseight (8) times more active in homing and penetrating into tumors thanthe original CGKRK-_(D)(KLAKLAK)₂ (SEQ ID NO: 8)-based nanosystemdescribed in, for example, U.S. Pat. Nos. 7,723,474 and 8,598,316, PCTInternational Patent Application Publication No. WO 2011/127405, andU.S. Patent Application Publication No. 2011/0262347. Thus, in someembodiments the TT1 Family of Peptides represented the lead peptidesgoing forward.

A goal of some embodiments of the methods disclosed herein was todevelop a novel targeted systems for selective delivery of therapeuticand diagnostic agents to GBM and other cancers. The original nanosystemin, for example, U.S. Pat. Nos. 7,723,474 and 8,598,316, PCTInternational Patent Application Publication No. WO 2001/127405, andU.S. Patent Application Publication No. 2011/0262347, employed threepeptides: CGKRK (SEQ ID NO: 6) as the homing peptide, a peptide known asiRGD to provide a tumor-penetrating function, and a pro-apoptoticpeptide, _(D)(KLAKLAK)₂ (SEQ ID NO: 42), as the “drug” (see also Agemyet al., 2011; 2013). The CGKRK (SEQ ID NO: 6) peptide specifically homesto angiogenic vasculature and enters into them. When capable ofaccessing tumor cells, it also binds to tumor cells of various types andinternalizes into them. However, CGKRK (SEQ ID NO: 6) lackstumor-penetrating activity, which confines CGKRK (SEQ ID NO: 6)-coatednanoparticles almost entirely to the vasculature (Joyce et al., 2003,Agemy et al., 2013), findings that were confirmed as described herein.

The LyP-1 Peptide is a cyclic 9-amino acid peptide that also uses p32 asits receptor (sequence: CGNKRTRGC (SEQ ID NO: 7); Laakkonen et al.,2002). Like CGKRK (SEQ ID NO: 6), LyP-1 internalizes into its targetcells, but like iRGD, LyP-1 is also capable of penetrating into tumorsand accumulating in the extravascular tumor tissue (Laakkonen et al.,2004; Roth et al. 2012). LyP-1 and iRGD contain a cryptic CendR (C-endRule) motif (R/KXXR/K), which binds to neuropilin-1 after the peptidehas been cleaved by a cell surface protease (Teesalu et al., 2009;Sugahara et al., 2009; 2010). The neuropilin-1 binding activates anendocytic pathway that transports payloads through the vascular wall andthrough tumor tissue, endowing these peptides with the tumor penetratingability. CGKRK (SEQ ID NO: 6) lacks the CendR function, and as aconsequence, it mainly targets tumor blood vessels.

As disclosed herein, two strategies were employed to simplify theoriginal nanosystem while also improving the homing and tumorpenetration properties of the system. In particular, a tumor-penetrating(CendR) activity was introduced into the peptide to facilitate thepenetration of the nanoparticles into the extracellular tumor tissuewithout the need of the separately injected iRGD peptide. To accomplishthese objectives, peptides that bound to the same receptor as CGKRK (SEQID NO: 6; i.e., p32/gC1qR/HABP) and contained the CendR motif wereidentified.

REFERENCES

All references listed in the instant disclosure, including but notlimited to all patents, patent applications, patent applicationpublications, scientific journal articles, and database entries(including but not limited to GENBANK® biosequence database entries andall annotations available therein) are incorporated herein by referencein their entireties to the extent not inconsistent herewith and to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

-   Abrahmsen et al. (1991) Biochemistry 30:4151.-   Agemy et al. (2011) Proc Natl Acad Sci USA 108:17450-17455.-   Agemy et al. (2013) Mol Ther 21:2195-2204.-   Alirol & Martinou (2006) Oncogene 25:4706-4716.-   Alitalo et al. (2004) Cancer Res 64:9225-9229.-   Allam et al. (1997) Cancer Res 57:2615-2618.-   Allen et al. (1979) Acta Crystallogr Section B, 35:2331-2339.-   Almquist et al. (1980) J Med Chem 23:1392-1398.-   Alvarez-Bravo et al. (1994) Biochem J 302:535-538.-   Arap et al. (1998a) Science 279:377-380.-   Arap et al. (1998b) 10:560-565.-   Arap et al. (2002) Proc Natl Acad Sci USA 99:1527-1531.-   Askew et al. (1989) JAm Chem Soc 111:1082-1090.-   Baggiolini al. (1992) FEBSLett 307:97-101.-   Bailey et al. (1980) Antimicrob Agents Chemother 17:549-553.-   Bangham et al. (1965) JMol Biol 13:238-252.-   Barenholz et al. (1979) FEBS Lett 99:210-214.-   Batzri et al. (1973) Biochim etBiophys Acta 298:1015-1019.-   Bessalle et al. (1990) FEBS Lett 274:151-155.-   Blancato et al. (2004) Br J Cancer 90:1612-1619.-   Blondelle & Houghten (1992a) Biochem 31:12688-12694.-   Blondelle & Houghten (1992b) in Annual Reports in Medicinal    Chemistry, Bristol (ed.), Academic Press, San Diego, Calif., United    States of America, pages 159-168.-   Bobak et al. (1987) J Immunol 138:1150-1156.-   Bobak et al. (1988) Eur J Immunol 18:2001-2007.-   Borgstrom et al. (1999) Anticancer Res 19:4213-4214.-   Braun et al. (2000) EMBO J 19:1458-1466.-   Brooks et al. (1994) JReprodMed 39:755-760.-   Callo et al. (1985) Cryobiology 22(3):251-267.-   Cane et al. (1998) Science 282:63-68.-   Chan et al. (1999)J Clin Oncol 17:2341-2354.-   Chen et al. (1994) J Immunol 153:1430-1440.-   Christian et al. (2003) J Cell Biol 163:871-878.-   Clark-Lewis et al. (1991) Biochemistry 30:3128.-   Clark-Lewis et al. (1994) J Biol Chem 269:16075.-   Creighton (1984) Proteins: Structures and Molecular Properties, W. H    Freeman, New York, N.Y., United States of America.-   Crooks et al. (2004) Genome Res 14:1188-1190.-   Davis et al. (1996) Cell 87:1161-1169.-   Dawson et al. (1994) Science 266:776-779.-   De Roos et al. (1991) IntlJ Card Imaging 7:133-138.-   Deamer et al. (1976) Biochim et Biophys Acta 443:629-634.-   Deb & Datta (1996) J Biol Chem 271:2206-2212.-   Dedio et al. (1998) J Immunol 160:3534-3542.-   Degenhardt et al. (2006) Cancer Cell 10:51-64. deLisle et al. (1992)    Techniques in Protein Chemistry IV. Academic Press, New York, N.Y.,    United States of America, pp. 257-267.-   Effert et al. (1996) J Urol 155:994-998.-   Ellerby (1999) Nature Med 5:1032-1038.-   European Patent Application Publication No. EP 0045665.-   Fantin et al. (2006) Cancer Cell 9:425-434.-   Ferrara et al. (1999) Nat Med 5:1359-1364.-   Ferreira et al. (2003) J Mol Catal B: Enzymatic 21:189-199.-   Fields et al. (1989) Nature 340:245-246.-   Fields et al. (1994) Trends Genet 10:286-292.-   Finlayson (1980) Semin Thromb Hemost, 6:85-120.-   Fisher et al. (1998) J Natl Cancer Inst 90:1371-1388.-   Fitzpatrick & Garnett (1995) Anticancer Drug Des 10:1-9.-   Fogal et al. (2008) Cancer Res 68:7210-7218.-   Fogal et al. (2010) Mol Cell Biol. 30: 1303-1318.-   Folkman & Shing (1992) J Biol Chem 267:10931-10934.-   Folkman (1997) Nature Biotechnol 15:510.-   Gao et al. (2002) J Biomed Opt 7:532.-   Garber (2006) Science 312:1158-1159.-   GENBANK® Biosequence Database Accession Nos. NM_001034527;    NM_001212; NM_007573; NM_019259; NP_001029699; NP_001203; NP_031599;    NP_062132; XM_001100940; XM_001918118; XM_002748302; XM_002826905;    XM_004058391; XM_005582637; XM_006939728; XM_546568; XP_001100940;    XP_001918153; XP_002748348; XP_002826951; XP_004058439;    XP_005582694; XP_006939790; XP_546568.-   Gerhardt et al. (1997) Eur J Pharmacol 15:334:1-23.-   German Patent Publication DE 1016978 19571003.-   Ghebrehiwet (1989) Behring Inst Mitt 84:204-215.-   Ghebrehiwet et al. (1994) J Exp Med 179:1809-1821.-   Ghebrehiwet et al. (2002) Immunobiology 205:421-432.-   Ghosh et al. (2004) Mol Cell Biochem 267:133-139.-   Goodman & Ro (1995) Peptidomimetics for Drug Design, in Burger's    Medicinal Chemistry and Drug Discovery Vol. 1, Wolff (ed), John    Wiley & Sons, New York, N.Y., United States of America, pages    803-861.-   Gregoriadis (1984) Liposome Technology, Vol. I-III, CRC Press, Boca    Raton, Fla., United States of America.-   Groziak (2001) Am J Therapeut 8:321-328.-   Guarino et al. (2004) Biotechnol Bioeng 86, 775-787.-   Guo et al. (1999) J Lab Clin Med 133:541-550.-   Gupta et al. (1991) Eur J Cell Biol 56:58-67.-   Hagedom & Bikfalvi (2000) Crit Rev OncolHematol 34:89-110.-   Hamzah et al. (2011) Proc Natl Acad Sci USA 108:7154-7159.-   Han et al. (2001) Nature Biotechnol 19:631.-   Hanahan & Weinberg (2000) Cell 100:57-70.-   Hann (1982) J Chem Soc Perkin Trans 1307-314.-   Harris (ed) (1992) Poly(ethylene glycol) Chemistry, Biotechnical and    Biomedical Applications, Plenum Press, New York, N.Y., United States    of America.-   Harris et al. (1997) in Cancer: Principles and Practice of Oncology    (5th ed.), DeVita et al. (eds) Lippincott Williams & Wilkins,    Philadelphia, Pa., United States of America.-   Haugland (2002) Handbook of Fluorescent Probes and Research Products    (9^(th) Edition), Molecular Probes, Inc., Eugene, Oreg., United    States of America.-   Henikoff & Henikoff (2000) Adv Protein Chem 54:73-97.-   Herwald et al. (1996) J Biol Chem 271:13040-13047.-   Hirasawa et al. (2001) Life Sci 68:2259-2267.-   Hodgson (1992) Bio/Technol 10:973-980.-   Hofer et al. (1999) Eur Urol 36:31-35.-   Hoffman et al. (2003) Cancer Cell 4:383-391.-   Holladay et al. (1983) Tetrahedron Lett 24:4401-4404.-   Homandberg et al. (1985) Am J Path 120:327-332.-   Homandberg et al. (1986) Biochim Biophys Acta 874:61-71.-   Houser et al. (1980) Surg Gynecol Obstet 150:811-816.-   Hruby (1982) Life Sci 31:189-199.-   Hruby et al. (1990) Biochem J 268:249-262.-   Hudson et al. (1979) Int J Pept Prot Res 14:177-185.-   Inoki et al. (2003) Cell 115:577-590.-   Isidoro et al. (2005) Carcinogenesis 26:2095-2104.-   Jaeger et al. (1989a) Methods Enzymol 183:281-306.-   Jaeger et al. (1989b) Proc Natl Acad Sci USA 86:7706-7710.-   Jain (1998) Nat Med 4:655-657.-   Jarvinen et al. (2007) Am J Pathol 171:702-711.-   Javadpour et al. (1996) J Med Chem 39:3107-3113.-   Jayawickreme et al. (1997) Curr Opin Biotechnol 8:629-634.-   Jennings-White et al. (1982) Tetrahedron Lett 23:2533-2534.-   Jiang et al. (1999) Proc Natl Acad Sci USA 96:3572-3577.-   Jin et al. (2007) J Cell Sci 120:379-383.-   Jones et al. (2005) Mol Cell 18:283-293.-   Joseph et al. (1996) Proc Natl Acad Sci USA 93:8552-8557.-   Joyce et al. (2003) Cancer Cell 4:393-403.-   Kaur et al. (1993) J Immunol 150:2046-2055.-   Kerj aschki (2005) J Clin Invest 115:2316-2319.-   Kerjaschki et al. (2006) Nat Med 12:230-234.-   Khan et al. (1998) Proc Natl Acad Sci USA 95:10425-10430.-   Kim et al. (1983) Biochim et Biophys Acta 728:339-348.-   Kirsch et al. (2000) J Neurooncol 50:149-163.-   Kittlesen et al. (2000) J Clin Invest 106:1239-1249.-   Koehler & Hess (1974) Biochemistry 13:5345-5350.-   Kohori et al. (1998) J Control Rel 55:87-98.-   Kohori et al. (1999) Colloids Surfaces B: Biointerfaces 16:195-205.-   Krainer et al. (1991) Cell 66:383-394.-   Kreitman & Pastan (1997) Blood 90:252-259.-   Kyte et al. (1982) J Mol Biol 157:105.-   Laakkonen et al. (2002) Nat Med 8:751-755.-   Laakkonen et al. (2004) Proc Natl Acad Sci USA 101:9381-9386.-   Lee et al. (2007) Mol Cancer Res 5:11-19.-   Leu et al. (1990) J Immunol 144:2281-2286.-   Levine (2007) Nature 446:745-747.-   Lewis & Dean (1989) Proc R Soc Lond 236:125-140 and 141-162.-   Liao et al. (2000) Endocr Relat Cancer 7:143-164.-   Liggins & Burt (2002) Adv Drug Del Rev 54:191-202.-   Lim et al. (1996) J Biol Chem 271:26739-26744.-   Lin et al. (2002) Appl Phys Lett 81:3134.-   Liu (2006) Prostate Cancer Prostatic Dis 9:230-234.-   Majumdar et al. (2002) Biochem Biophys Res Commun 291:829-837.-   Maloy & Kari (1995) Biopolymers 37:105-122.-   Maloy et al. (1995) Biopolymers 37:105-122.-   Mancheno et al. (1998) J Peptide Res 51:142-148.-   Martin et al. (2000) Cancer Res 60:3218-3224.-   Maruyama et al. (2005)J Clin Invest 115:2363-2372.-   Maruyama et al. (2007) Am J Pathol 170:1178-1191.-   Matthews & Russell (1998) J Gen Virol 79 (Pt 7): 1677-1685.-   McKinaly & Rossmann (1989) Ann Rev Pharmacol Toxiciol 29:111-122.-   Moghimi et al. (2001) Pharm Rev 53:283-318.-   Moghimi et al. (2001) Pharmacol Rev 53:283-318.-   Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243-252.-   Morley (1980) Trends Pharmacol Sci 1:463-468.-   Muta et al. (1997) J Biol Chem 272:24363-24370.-   Myers (1997) Curr Opin Biotechnol 8:701-707.-   Needleman & Wunsch (1970) J Mol Biol 48:443.-   Oh et al. (2004) Nature 429:629-635.-   O'Reilly et al. (1994) Cell 79:315-328.-   O'Reilly et al. (1997) Cell 88:277-285.-   O'Reilly et al. (1999) Science 285:1926-1928.-   Osborne & Coronado-Heinsohn (1996) Cancer J Sci Am 2:175-180.-   Pai et al. (1999) Mag Magnet Mater 194:262.-   Papahadj opoulos et al. (1968) Biochim et Biophys Acta 135:624-238.-   Paridaens et al. (2000) J Clin Oncol 18:724-733.-   Park et al. (2008) Adv Mater 20:1630-1635.-   Parle-McDermott et al. (2000) Br J Cancer 83:725-728.-   Pasqualini et al (2000) Cancer Res 60:722-727.-   Pausch (1997) Trends Biotech 15:487-494.-   PCT International Patent Application Publication Nos. WO 96/32434;    WO 96/33233; WO 97/00623; WO 98/37177; WO 99/64446; WO 2002/044184;    WO 2011/127405.-   PCT International Patent Application Serial No. PCT/US2011/31785.-   Pearson & Lipman (1988) Proc Natl Acad Sci USA 85:2444.-   Peerschke et al. (1993) JExp Med 178:579-587.-   Peerschke et al. (1994) J Immunol 152:5896-5901.-   Perry & Davies (1989) QSAR: Quantitative Structure-Activity    Relationships. in Drug Design, Alan R. Liss, Inc., New York, N.Y.,    United States of America, pages 189-193.-   Pilch et al. (2006) Proc Natl Acad Sci USA 103:2800-2804.-   Pirollo et al. (2007) Cancer Res 67:2938-2943.-   Porkka et al. (2002) Proc Natl Acad Sci USA 99:7444-7449.-   Powers et al. (1982) Neurology 32: 938.-   Raj arathnam et al. (1994) Biochemistry 33:6623-6630).-   Reef et al. (2007) Oncogene 26:6677-6683.-   Remington (1995) The Science and Practice of Pharmacy (19th ed.)    Gennaro (ed.), Mack Publishing Company, Easton, Pa., United States    of America.-   Ripka (1988), New Scientist 54-57 (Jun. 16, 1988).-   Robles-Flores et al. (2002) J Biol Chem 277:5247-5255.-   Roth et al. (2012) Transtumoral targeting enabled by a novel    neuropilin-binding peptide. Oncogene 31:3754-3763.-   Rotivinen et al. (1988) Acta Pharmaceutica Fennica 97:159-166.-   Rozanov et al. (2002a) J Biol Chem 277:9318-9325.-   Rozanov et al. (2002b) FEBS Lett 527:51-57.-   Rubinstein et al. (2004) Intl J Cancer 110:741-750.-   Rubinsztein et al. (2007) Nat Rev Drug Discov 6:304-312.-   Ruoslahti (2002) Nat Rev Cancer 2:83-90.-   Ruoslahti et al. (2010) J Cell Biology 188:759-768.-   Rusinko et al. (1989) J Chem Inf Comput Sci 29:251-255.-   Saberwal et al. (1994) Biochim Biophys Acta 1197:109-131.-   Saharinen et al. (2004) Trends Immunol 25:387-395.-   Schaerer et al. (2001) J Biol Chem 276:26597-26604.-   Schledzewski et al. (2006) J Pathol 209:67-77.-   Schneider & Stephens (1990) Nucleic Acids Res 18:6097-6100.-   Schnolzer et al. (1992) Science 256:221.-   Schroeder et al. (1996) J Biomol Screening 1:75-80.-   Sengupta et al. (2004) Biochem J 380:837-844.-   Shaw (2006) Curr Opin Cell Biol 18:598-608.-   Shim et al. (1997) Proc Natl Acad Sci USA 94:6658-6663.-   Sim & Reid (1991) Immunol Today 12:307-311.-   Simberg et al. (2007) Proc Natl Acad Sci USA 104:932-936.-   Simpelkamp & Jones (1992) Bioorg Med Chem Lett 2:1391-1394.-   Singh et al. (1997) Exp Cell Res 234:205-216.-   Slavin et al. (1995) Cell Biol Intl 19:431-444.-   Smith & Waterman (1981) Adv Appl Math 2:482.-   Soltys & Gupta (1996) Exp Cell Res 222:16-27.-   Soltys & Gupta (1997) Cell Biol Intl 21:315-320.-   Soltys & Gupta (1999) Trends Biochem Sci 24:174-177.-   Soltys et al. (2000) Histochem Cell Biol 114:245-255.-   Spatola (1983a) Chemistry and Biochemistry of Amino Acids, Peptides,    and Proteins (Weinstein eds.), Marcel Dekker, New York, N.Y., United    States of America, page 267.-   Spatola (1983b) Peptide Backbone Modifications, Vega Data 1(3).-   Spatola et al. (1986) Life Sci 38:1243-1249.-   St. Croix et al. (2000) Science 289:1197-1202.-   Stacker et al. (2002) Nat Rev Cancer 2:573-583.-   Steiner et al. (eds.) (1992) in Angiogenesis: Key    Principles—Science, Technology, Medicine Birkhauser Verlag, Boston,    Mass., United States of America, pp. 449-454.-   Stewart & Ratain (1997) in Cancer: Principles and Practice of    Oncology (5th ed.), DeVita et al. (eds) Lippincott Williams &    Wilkins, Philadelphia, Pa., United States of America.-   Storz et al. (2000) J Biol Chem 275:24601-24607.-   Strosberg et al. (1992) Trends Pharm Sci 13:95-98.-   Sugahara et al. (2009) Cancer Cell 16:510-520.-   Sugahara et al. (2010) Science 328:1031-1035.-   Suri et al. (1996) Cell 87:1171-1180.-   Sweetnam et al. (1993) J Nat Prod 56:441-455.-   Tange et al. (1996) J Biol Chem 271:10066-10072.-   Teesalu et al. (2009) Proc Natl Acad Sci USA 106:16157-16162.-   Thakur et al. (1976) Throm Res 9:345.-   Tullis (1977) J Am Med Assoc 237:355-360, 460-463.-   Tuzar & Kratochvil (1976) Adv Colloid Interface Sci 6:201-232.-   U.S. Patent Application Publication No. 2004/0009122; 2004/0087499;    2005/0004002; 2007/0219134; 2008/0014143; 2008/0305101;    2009/0036349; 2009/0226372; 2010/0322862; 2011/0262347.-   U.S. patent application Ser. No. 08/996,783.-   U.S. Pat. Nos. 4,016,100; 4,089,801; 4,235,871; 4,418,052;    4,485,054; 4,554,101; 4,745,160; 4,761,288; 4,853,228; 5,011,686;    5,013,497; 5,024,829; 5,410,016; 5,412,072; 5,449,513; 5,474,848;    5,534,499; 5,585,277; 5,628,936; 5,693,751; 5,789,542; 5,792,742;    5,820,873; 5,885,613; 5,891,646; 5,897,945; 5,906,820; 5,916,596;    5,925,720; 5,929,177; 6,110,693; 6,320,017; 6,506,405; 6,530,944;    6,537,579; 6,759,199; 7,544,767; 7,723,474; 8,367,621; 8,598,316.-   van Leeuwen & O'Hare (2001) J Cell Sci 114:2115-2123.-   Van Rooijen & Sanders (1994) J Immunol Meth 174:83-93.-   Vanden Broeck (1996) Intl Rev Cytol 164:189-268.-   Waggoner et al. (2005) J Immunol 175:4706-4714.-   Wallace (2005) Cold Spring Harb Symp Quant Biol 70:363-374.-   Wang et al. (2003) Chem Mater 15:2724.-   Weder et al. (1984) in Liposome Technology, Gregoriadis (ed.), CRC    Press Inc., Boca Raton, Fla., United States of America, Vol. I,    Chapter 7, pages 79-107.-   Weinand et al. (1999) Bioorg Med Chem 7:1295-1307.-   White et al. (2001) Ann Rev Med 52:125-141.-   Wieboldt et al. (1997) Anal Chem 69:1683-1691.-   Wilhelm et al. (1991) Macromolecules 24:1033-1040.-   Williams (1991) Med Res Rev 11:147-184.-   Wilson et al. (1998) Brit J Pharmacol 125:1387-1392.-   Xu et al. (1994) Circ Res 75:1078-1085.-   Yagi et al. (2012) Nucl. Acids Res. 40: 9717-9737.-   Yamamoto (1991) Pure Appl Chem 63:423-426.-   Young et al. (1991) J Immunol 146:3356-3364.-   Zhang et al. (1996) Intl J Pharm 132:195-206.-   Zhang et al. (2006) Cancer Res 66:5696-5706.-   Zhang et al. (2007) Autophagy 3:337-346.-   Zuker (1989) Science 244:48-52.

It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

1. An isolated peptide or peptidomimetic comprising the amino acidsequence KRGARST (SEQ ID NO: 1) or a peptidomimetic thereof; the aminoacid sequence AKRGARSTA (SEQ ID NO: 2) or a peptidomimetic thereof; or acombination thereof, wherein the isolated peptide or peptidomimetic is alinear peptide or peptidomimetic. 2-7. (canceled)
 8. The isolatedpeptide or peptidomimetic of claim 1, which has a length of less than 50residues. 9-10. (canceled)
 11. A conjugate comprising one or moremoieties linked to a homing molecule that selectively homes to tumorsand penetrates into extravascular tumor tissue, wherein the homingmolecule comprises an isolated peptide or peptidomimetic of claim
 1. 12.The conjugate of claim 11, wherein the conjugate further comprises oneor more additional homing molecules comprising an antibody orantigen-binding fragment thereof that binds to an antiqen present in atumor.
 13. The conjugate of claim 11, wherein the homing molecule is apeptide comprising SEQ ID NO: 1 or SEQ ID NO:
 2. 14. (canceled)
 15. Theconjugate of claim 13, wherein the peptide has a length of at most 50residues. 16-25. (canceled)
 26. The conjugate of claim 11, wherein theone or more moieties comprises a therapeutic agent, a detectable agent,or a combination thereof. 27-91. (canceled)
 92. A composition thatcomprises a surface molecule, one or more homing molecules, and aplurality of membrane perturbing molecules, wherein at least one of theone or more homing molecules comprises the amino acid sequence KRGARST(SEQ ID NO: 1), and further wherein the isolated peptide orpeptidomimetic is a linear peptide or peptidomimetic that selectivelyhomes to a tumor. 93-119. (canceled)
 120. The composition of claim 92,wherein one or more of the homing molecules comprise one or more of theinternalization elements. 121-122. (canceled)
 123. The composition ofclaim 92, wherein the composition further comprises one or more tissuepenetration elements. 124-129. (canceled)
 130. The composition of claim92, wherein the surface molecule is selected from the group consistingof a nanoparticle, a liposome, a micelle, and a microparticle. 131.(canceled)
 132. The composition of claim 130, wherein the surfacemolecule is selected from the group consisting of an iron oxidenanoparticle and an albumin nanoparticle. 133-153. (canceled)
 154. Thecomposition of claim 92, further comprising one or more therapeuticmoieties, one or more detectable moieties, or any combination thereof.155. The composition of claim 154, wherein the one or more therapeuticmoieties and the one or more detectable moieties are independentlyselected from the group consisting of an anti-angiogenic agent, apro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxicagent, an anti-inflammatory agent, an anti-arthritic agent, apolypeptide, a nucleic acid molecule, a small molecule, an imagecontrast agent, a fluorophore, fluorescein, rhodamine, a radionuclide,indium-111, technetium-99, carbon-11, and carbon-13. 156-178. (canceled)179. A method for detecting the presence of a gC1q/p32 receptor, themethod comprising: (a) bringing into contact a cell and a compositioncomprising the isolated peptide or peptidomimetic of claim 1 conjugatedto a detectable agent; and (b) detecting an interaction between thegC1q/p32 receptor and the isolated peptide or peptidomimetic of claim 1,thereby detecting the presence of gC1q/p32 receptor. 180-192. (canceled)193. A method for identifying a subject having a disease or disorderassociated with a gC1q/p32 receptor biological activity, the methodcomprising: (a) bringing into contact a cell of the subject and acomposition comprising the isolated peptide or peptidomimetic of claim 1conjugated to a detectable agent; and (b) detecting interaction betweengC1q/p32 receptor and the composition, thereby detecting the presence orlevel of gC1q/p32, wherein the presence or level of gC1q/p32 receptoridentifies the subject as having a disease or disorder associated with agC1q/p32 receptor biological activity. 194-199. (canceled)